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Finding an accurate and affordable method to quantify muscle size following spinal cord injury (SCI) could provide benefits clinically and in research settings. The purpose of this study was to validate the use of anthropometric measurements vs. magnetic resonance imaging (MRI) to evaluate muscle cross-sectional area (CSA) and develop a field equation to predict muscle CSA specific to the SCI population. Twenty-two men with chronic (>1 yr) motor complete SCI participated in the current study. Anthropometric measurements, including midthigh circumference and anterior skinfold thickness (SF T ), were taken on the right thigh. The anthropometric muscle cross-sectional area (muscle CSA anthro ) was predicted using the following equation: muscle CSA anthro = π[ r - (SF T /2)] 2 , where r = thigh circumference/2π. MRI analysis yielded whole thigh CSA (thigh CSA MRI ), midthigh muscle CSA (muscle CSA MRI ), midthigh absolute muscle CSA after subtracting intramuscular fat and bone (muscle CSA-IMF MRI ), subcutaneous adipose tissue (SAT T ) measured at one site as well as at four sites, and bone CSA. Anthropometric measurements were correlated to the thigh CSA MRI [ r 2 = 0.90, standard error of the estimate (SEE) = 17.6 cm 2 , P < 0.001]. Muscle CSA anthro was correlated to muscle CSA MRI ( r 2 = 0.78, SEE = 16.6 cm 2 , P < 0.001) and muscle CSA-IMF MRI ( r 2 = 0.75, SEE = 17.6 cm 2 , P < 0.001). A single SF T was correlated to the polar four-site SAT T ( r 2 = 0.78, SEE = 0.37 cm, P < 0.001). The average femur CSA and average IMF CSA derived from MRI led to the following field equation: muscle CSA predicted = π[(Thigh circum /2π) - (SF T /2)] 2 - 23.2. Anthropometric measurements of muscle CSA exhibited a good agreement with the gold standard MRI method and led to the development of a field equation for clinical use after accounting for bone and IMF. NEW & NOTEWORTHY This study used anthropometric measurements and magnetic resonance imaging (MRI) to evaluate muscle cross-sectional area (CSA) and developed a field equation to predict thigh muscle CSA specific to the spinal cord-injured (SCI) population. Anthropometric measurements were correlated to the whole thigh CSA and muscle CSA as measured by MRI . The correlations led to the development of a SCI-specific field equation that accounted for intramuscular fat and bone areas.

Flavonoids have previously been identified as antiparasitic agents and pteridine reductase 1 (PTR1) inhibitors. Herein, we focus our attention on the chroman-4-one scaffold. Three chroman-4-one analogues ( 1 - 3 ) of previously published chromen-4-one derivatives were synthesized and biologically evaluated against parasitic enzymes ( Trypanosoma brucei PTR1- Tb PTR1 and Leishmania major-Lm PTR1) and parasites ( Trypanosoma brucei and Leishmania infantum ). A crystal structure of Tb PTR1 in complex with compound 1 and the first crystal structures of Lm PTR1-flavanone complexes (compounds 1 and 3 ) were solved. The inhibitory activity of the chroman-4-one and chromen-4-one derivatives was explained by comparison of observed and predicted binding modes of the compounds. Compound 1 showed activity both against the targeted enzymes and the parasites with a selectivity index greater than 7 and a low toxicity. Our results provide a basis for further scaffold optimization and structure-based drug design aimed at the identification of potent anti-trypanosomatidic compounds targeting multiple PTR1 variants.

Spinal cord injury (SCI) is accompanied by deterioration in body composition and severe muscle atrophy. These changes put individuals at risk for insulin resistance, type II diabetes, and cardiovascular disease. To determine the relationships between skeletal muscle mitochondrial mass, activity, and body composition, 22 men with motor complete SCI were studied. Body composition assessment was performed using dual-energy X-ray absorptiometry and magnetic resonance imaging. Skeletal muscle biopsies were obtained from the vastus lateralis muscle to measure citrate synthase (CS) and complex III (CIII) activity. CS activity was inversely related to %body fat ( r  = -0.57, P  = 0.013), %leg fat ( r  = -0.52, P  = 0.027), %trunk fat ( r  = -0.54, P  = 0.020), and %android fat ( r  = -0.54, P  = 0.017). CIII activity was negatively related to %body fat ( r  = -0.58, P  = 0.022) and %leg fat ( r  = -0.54, P  = 0.037). Increased visceral adipose tissue was associated with decreased CS and CIII activity ( r  = -0.66, P  = 0.004; r  = -0.60, P  = 0.022). Thigh intramuscular fat was also inversely related to both CS and CIII activity ( r  = -0.56, P  = 0.026; r  = -0.60, P  = 0.024). Conversely, lean mass ( r  = 0.75, P  = 0.0003; r  = 0.65, P  = 0.008) and thigh muscle cross-sectional area (CSA; r  = 0.82, P  = 0.0001; r  = 0.84; P  = 0.0001) were positively related to mitochondrial parameters. When normalized to thigh muscle CSA, many body composition measurements remained related to CS and CIII activity, suggesting that %fat and lean mass may predict mitochondrial mass and activity independent of muscle size. Finally, individuals with SCI over age 40 had decreased CS and CIII activity ( P  = 0.009; P  = 0.004), suggesting a decrease in mitochondrial health with advanced age. Collectively, these findings suggest that an increase in adipose tissue and decrease in lean mass results in decreased skeletal muscle mitochondrial activity in individuals with chronic SCI., (© 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

The enzyme adenylyl cyclase (AC) plays a pivotal role in a variety of signal transduction pathways inside the cell, where it catalyzes the cyclization of adenosine triphosphate (ATP) into the second-messenger cyclic adenosine monophosphate (cAMP). Among other roles, AC regulates processes involved in neural plasticity, innervation of smooth muscles of the heart and the endocrine system of the pancreas. The functional diversity of AC is manifested in its different isoforms, each having a specific regulation pattern. There is an increasing amount of data available concerning the regulatory properties of AC isoforms, however little is known about the interactions on a structural level. Here, we conducted a comparative electrostatic analysis of the catalytic domains of all nine transmembrane AC isoforms with the aim of detecting, verifying and predicting the binding sites of molecular regulators on AC. The results provide support for the positioning of the binding site of the inhibitory protein G i α at a pseudo-symmetric position to the stimulatory G s α binding site. They also provide a structural interpretation of the Gβγ interaction with ACs 2, 4, and 7 and suggest a new binding site for RGS2. Comparison of the small molecule binding sites on AC shows that overall they have high electrostatic similarity, but regions of electrostatic differences are identified. These could provide a basis for the development of novel compounds with isoform-specific modulatory effects on AC. Proteins 2016; 84:1844-1858. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Computer simulations of molecular systems often make use of regular rectangular grids with equidistant spacing to store information on their molecular interaction fields, e.g., electrostatic potential. These grids provide an easy way to store the data as they do not require any particular specification of the structure of the data. However, such grids may easily become large, and the storage and memory demands may become so high that calculations become infeasible. To overcome this problem, we show here how the data structure DT-Grid can be adapted and applied to efficiently represent macromolecular interaction grids by exploiting the nonuniformity of information on the grid; at the same time, this data structure ensures fast random data access. We demonstrate use of the DT-Grid data structure for potential of mean force and Brownian dynamics simulations of protein-surface binding and macromolecular association with the SDA software. We further demonstrate that the DT-Grid structure enables systems of large size, such as a viral capsid, and high resolution grids to be handled that are beyond current computational feasibility.

We present the results for CAPRI Round 30, the first joint CASP-CAPRI experiment, which brought together experts from the protein structure prediction and protein-protein docking communities. The Round comprised 25 targets from amongst those submitted for the CASP11 prediction experiment of 2014. The targets included mostly homodimers, a few homotetramers, and two heterodimers, and comprised protein chains that could readily be modeled using templates from the Protein Data Bank. On average 24 CAPRI groups and 7 CASP groups submitted docking predictions for each target, and 12 CAPRI groups per target participated in the CAPRI scoring experiment. In total more than 9500 models were assessed against the 3D structures of the corresponding target complexes. Results show that the prediction of homodimer assemblies by homology modeling techniques and docking calculations is quite successful for targets featuring large enough subunit interfaces to represent stable associations. Targets with ambiguous or inaccurate oligomeric state assignments, often featuring crystal contact-sized interfaces, represented a confounding factor. For those, a much poorer prediction performance was achieved, while nonetheless often providing helpful clues on the correct oligomeric state of the protein. The prediction performance was very poor for genuine tetrameric targets, where the inaccuracy of the homology-built subunit models and the smaller pair-wise interfaces severely limited the ability to derive the correct assembly mode. Our analysis also shows that docking procedures tend to perform better than standard homology modeling techniques and that highly accurate models of the protein components are not always required to identify their association modes with acceptable accuracy. Proteins 2016; 84(Suppl 1):323-348. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Sequence-specific cleavage of collagen by mammalian collagenase plays a pivotal role in cell function. Collagenases are matrix metalloproteinases that cleave the peptide bond at a specific position on fibrillar collagen. The collagenase Hemopexin-like (HPX) domain has been proposed to be responsible for substrate recognition, but the mechanism by which collagenases identify the cleavage site on fibrillar collagen is not clearly understood. In this study, Brownian dynamics simulations coupled with atomic-detail and coarse-grained molecular dynamics simulations were performed to dock matrix metalloproteinase-1 (MMP-1) on a collagen IIIα1 triple helical peptide. We find that the HPX domain recognizes the collagen triple helix at a conserved R-X11-R motif C-terminal to the cleavage site to which the HPX domain of collagen is guided electrostatically. The binding of the HPX domain between the two arginine residues is energetically stabilized by hydrophobic contacts with collagen. From the simulations and analysis of the sequences and structural flexibility of collagen and collagenase, a mechanistic scheme by which MMP-1 can recognize and bind collagen for proteolysis is proposed.

Flavonoids represent a potential source of new antitrypanosomatidic leads. Starting from a library of natural products, we combined target-based screening on pteridine reductase 1 with phenotypic screening on Trypanosoma brucei for hit identification. Flavonols were identified as hits, and a library of 16 derivatives was synthesized. Twelve compounds showed EC50 values against T. brucei below 10 μM. Four X-ray crystal structures and docking studies explained the observed structure-activity relationships. Compound 2 (3,6-dihydroxy-2-(3-hydroxyphenyl)-4H-chromen-4-one) was selected for pharmacokinetic studies. Encapsulation of compound 2 in PLGA nanoparticles or cyclodextrins resulted in lower in vitro toxicity when compared to the free compound. Combination studies with methotrexate revealed that compound 13 (3-hydroxy-6-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one) has the highest synergistic effect at concentration of 1.3 μM, 11.7-fold dose reduction index and no toxicity toward host cells. Our results provide the basis for further chemical modifications aimed at identifying novel antitrypanosomatidic agents showing higher potency toward PTR1 and increased metabolic stability.

Linker histones are essential for DNA compaction in chromatin. They bind to nucleosomes in a 1:1 ratio forming chromatosomes. Alternative configurations have been proposed in which the globular domain of the linker histone H5 (gH5) is positioned either on- or off-dyad between the nucleosomal and linker DNAs. However, the dynamic pathways of chromatosome assembly remain elusive. Here, we studied the conformational plasticity of gH5 in unbound and off-dyad nucleosome-bound forms with classical and accelerated molecular dynamics simulations. We find that the unbound gH5 converts between open and closed conformations, preferring the closed form. However, the open gH5 contributes to a more rigid chromatosome and restricts the motion of the nearby linker DNA through hydrophobic interactions with thymidines. Moreover, the closed gH5 opens and reorients in accelerated simulations of the chromatosome. Brownian dynamics simulations of chromatosome assembly, accounting for a range of amplitudes of nucleosome opening and different nucleosome DNA sequences, support the existence of both on- and off-dyad binding modes of gH5 and reveal alternative, sequence and conformation-dependent chromatosome configurations. Taken together, these findings suggest that the conformational dynamics of linker histones and nucleosomes facilitate alternative chromatosome configurations through an interplay between induced fit and conformational selection., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Signal recognition particle (SRP) is a universally conserved protein-RNA complex that mediates co-translational protein translocation and membrane insertion by targeting translating ribosomes to membrane translocons. The existence of parallel co- and post-translational transport pathways, however, raises the question of the cellular substrate pool of SRP and the molecular basis of substrate selection. Here we determine the binding sites of bacterial SRP within the nascent proteome of Escherichia coli at amino acid resolution, by sequencing messenger RNA footprints of ribosome-nascent-chain complexes associated with SRP. SRP, on the basis of its strong preference for hydrophobic transmembrane domains (TMDs), constitutes a compartment-specific targeting factor for nascent inner membrane proteins (IMPs) that efficiently excludes signal-sequence-containing precursors of periplasmic and outer membrane proteins. SRP associates with hydrophobic TMDs enriched in consecutive stretches of hydrophobic and bulky aromatic amino acids immediately on their emergence from the ribosomal exit tunnel. By contrast with current models, N-terminal TMDs are frequently skipped and TMDs internal to the polypeptide sequence are selectively recognized. Furthermore, SRP binds several TMDs in many multi-spanning membrane proteins, suggesting cycles of SRP-mediated membrane targeting. SRP-mediated targeting is not accompanied by a transient slowdown of translation and is not influenced by the ribosome-associated chaperone trigger factor (TF), which has a distinct substrate pool and acts at different stages during translation. Overall, our proteome-wide data set of SRP-binding sites reveals the underlying principles of pathway decisions for nascent chains in bacteria, with SRP acting as the dominant triaging factor, sufficient to separate IMPs from substrates of the SecA-SecB post-translational translocation and TF-assisted cytosolic protein folding pathways.

Simulations of the long-time scale motions of a ligand binding pocket in a protein may open up new perspectives for the design of compounds with steric or chemical properties differing from those of known binders. However, slow motions of proteins are difficult to access using standard molecular dynamics (MD) simulations and are thus usually neglected in computational drug design. Here, we introduce two nonequilibrium MD approaches to identify conformational changes of a binding site and detect transient pockets associated with these motions. The methods proposed are based on the rotamerically induced perturbation (RIP) MD approach, which employs perturbation of side-chain torsional motion for initiating large-scale protein movement. The first approach, Langevin-RIP (L-RIP), entails a series of short Langevin MD simulations, each starting with perturbation of one of the side-chains lining the binding site of interest. L-RIP provides extensive sampling of conformational changes of the binding site. In less than 1 ns of MD simulation with L-RIP, we observed distortions of the α-helix in the ATP binding site of HSP90 and flipping of the DFG loop in Src kinase. In the second approach, RIPlig, a perturbation is applied to a pseudoligand placed in different parts of a binding pocket, which enables flexible regions of the binding site to be identified in a small number of 10 ps MD simulations. The methods were evaluated for four test proteins displaying different types and degrees of binding site flexibility. Both methods reveal all transient pocket regions in less than a total of 10 ns of simulations, even though many of these regions remained closed in 100 ns conventional MD. The proposed methods provide computationally efficient tools to explore binding site flexibility and can aid in the functional characterization of protein pockets, and the identification of transient pockets for ligand design.

PRO: tein S: tructure A: nnotation T: ool-plus (ProSAT(+)) is a new web server for mapping protein sequence annotations onto a protein structure and visualizing them simultaneously with the structure. ProSAT(+) incorporates many of the features of the preceding ProSAT and ProSAT2 tools but also provides new options for the visualization and sharing of protein annotations. Data are extracted from the UniProt KnowledgeBase, the RCSB PDB and the PDBe SIFTS resource, and visualization is performed using JSmol. User-defined sequence annotations can be added directly to the URL, thus enabling visualization and easy data sharing. ProSAT(+) is available at http://prosat.h-its.org., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The signal recognition particle (SRP) is a ribonucleoprotein complex with a key role in targeting and insertion of membrane proteins. The two SRP GTPases, SRP54 (Ffh in bacteria) and FtsY (SRα in eukaryotes), form the core of the targeting complex (TC) regulating the SRP cycle. The architecture of the TC and its stimulation by RNA has been described for the bacterial SRP system while this information is lacking for other domains of life. Here, we present the crystal structures of the GTPase heterodimers of archaeal (Sulfolobus solfataricus), eukaryotic (Homo sapiens), and chloroplast (Arabidopsis thaliana) SRP systems. The comprehensive structural comparison combined with Brownian dynamics simulations of TC formation allows for the description of the general blueprint and of specific adaptations of the quasi-symmetric heterodimer. Our work defines conserved external nucleotide-binding sites for SRP GTPase activation by RNA. Structural analyses of the GDP-bound, post-hydrolysis states reveal a conserved, magnesium-sensitive switch within the I-box. Overall, we provide a general model for SRP cycle regulation by RNA., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The dynamics of protein binding pockets are crucial for their interaction specificity. Structural flexibility allows proteins to adapt to their individual molecular binding partners and facilitates the binding process. This implies the necessity to consider protein internal motion in determining and predicting binding properties and in designing new binders. Although accounting for protein dynamics presents a challenge for computational approaches, it expands the structural and physicochemical space for compound design and thus offers the prospect of improved binding specificity and selectivity. A cavity on the surface or in the interior of a protein that possesses suitable properties for binding a ligand is usually referred to as a binding pocket. The set of amino acid residues around a binding pocket determines its physicochemical characteristics and, together with its shape and location in a protein, defines its functionality. Residues outside the binding site can also have a long-range effect on the properties of the binding pocket. Cavities with similar functionalities are often conserved across protein families. For example, enzyme active sites are usually concave surfaces that present amino acid residues in a suitable configuration for binding low molecular weight compounds. Macromolecular binding pockets, on the other hand, are located on the protein surface and are often shallower. The mobility of proteins allows the opening, closing, and adaptation of binding pockets to regulate binding processes and specific protein functionalities. For example, channels and tunnels can exist permanently or transiently to transport compounds to and from a binding site. The influence of protein flexibility on binding pockets can vary from small changes to an already existent pocket to the formation of a completely new pocket. Here, we review recent developments in computational methods to detect and define binding pockets and to study pocket dynamics. We introduce five different classes of protein pocket dynamics: (1) appearance/disappearance of a subpocket in an existing pocket; (2) appearance/disappearance of an adjacent pocket on the protein surface in the direct vicinity of an already existing pocket; (3) pocket breathing, which may be caused by side-chain fluctuations or backbone or interdomain vibrational motion; (4) opening/closing of a channel or tunnel, connecting a pocket inside the protein with solvent, including lid motion; and (5) the appearance/disappearance of an allosteric pocket at a site on a protein distinct from an already existing pocket with binding of a ligand to the allosteric binding site affecting the original pocket. We suggest that the class of pocket dynamics, as well as the type and extent of protein motion affecting the binding pocket, should be factors considered in choosing the most appropriate computational approach to study a given binding pocket. Furthermore, we examine the relationship between pocket dynamics classes and induced fit, conformational selection, and gating models of ligand binding on binding kinetics and thermodynamics. We discuss the implications of protein binding pocket dynamics for drug design and conclude with potential future directions for computational analysis of protein binding pocket dynamics.

The addition of three N-terminal histidines to β-lactamase inhibitor protein was shown experimentally to increase its binding potency to an Au(111) surface substantially but the binding mechanism was not resolved. Here, we propose a complete adsorption mechanism for this fusion protein by means of a multi-scale simulation approach and free energy calculations. We find that adsorption is a three-step process: (i) recognition of the surface predominantly by the histidine fusion peptide and formation of an encounter complex facilitated by a reduced dielectric screening of water in the interfacial region, (ii) adsorption of the protein on the surface and adoption of a specific binding orientation, and (iii) adaptation of the protein structure on the metal surface accompanied by induced fit. We anticipate that the mechanistic features of protein adsorption to an Au(111) surface revealed here can be extended to other inorganic surfaces and proteins and will therefore aid the design of specific protein-surface interactions.

The high-mobility group box 1 (HMGB1) protein has a central role in immunological antitumour defense. Here we show that natural killer cell-derived HMGB1 directly eliminates cancer cells by triggering metabolic cell death. HMGB1 allosterically inhibits the tetrameric pyruvate kinase isoform M2, thus blocking glucose-driven aerobic respiration. This results in a rapid metabolic shift forcing cells to rely solely on glycolysis for the maintenance of energy production. Cancer cells can acquire resistance to HMGB1 by increasing glycolysis using the dimeric form of PKM2, and employing glutaminolysis. Consistently, we observe an increase in the expression of a key enzyme of glutaminolysis, malic enzyme 1, in advanced colon cancer. Moreover, pharmaceutical inhibition of glutaminolysis sensitizes tumour cells to HMGB1 providing a basis for a therapeutic strategy for treating cancer.

Background: Intellectual disability (ID) is a neurodevelopmental disorder affecting 1%-3% of the population worldwide. It is characterised by high phenotypic and genetic heterogeneity and in most cases the underlying cause of the disorder is unknown. In our study we investigated a large consanguineous family from Baluchistan, Pakistan, comprising seven affected individuals with a severe form of autosomal recessive ID (ARID) and epilepsy, to elucidate a putative genetic cause., Methods and Results: Whole exome sequencing (WES) of a trio, including a child with ID and epilepsy and its healthy parents that were part of this large family, revealed a homozygous missense variant p.R53Q in the lectin mannose-binding 2-like (LMAN2L) gene. This homozygous variant was co-segregating in the family with the phenotype of severe ID and infantile epilepsy; unaffected family members were heterozygous variant carriers. The variant was predicted to be pathogenic by five different in silico programmes and further three-dimensional structure modelling of the protein suggests that variant p.R53Q may impair protein-protein interaction. LMAN2L (OMIM: 609552) encodes for the lectin, mannose-binding 2-like protein which is a cargo receptor in the endoplasmic reticulum important for glycoprotein transport. Genome-wide association studies have identified an association of LMAN2L to different neuropsychiatric disorders., Conclusion: This is the first report linking LMAN2L to a phenotype of severe ARID and seizures, indicating that the deleterious homozygous p.R53Q variant very likely causes the disorder., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Understanding protein-inorganic surface interactions is central to the rational design of new tools in biomaterial sciences, nanobiotechnology and nanomedicine. Although a significant amount of experimental research on protein adsorption onto solid substrates has been reported, many aspects of the recognition and interaction mechanisms of biomolecules and inorganic surfaces are still unclear. Theoretical modeling and simulations provide complementary approaches for experimental studies, and they have been applied for exploring protein-surface binding mechanisms, the determinants of binding specificity towards different surfaces, as well as the thermodynamics and kinetics of adsorption. Although the general computational approaches employed to study the dynamics of proteins and materials are similar, the models and force-fields (FFs) used for describing the physical properties and interactions of material surfaces and biological molecules differ. In particular, FF and water models designed for use in biomolecular simulations are often not directly transferable to surface simulations and vice versa. The adsorption events span a wide range of time- and length-scales that vary from nanoseconds to days, and from nanometers to micrometers, respectively, rendering the use of multi-scale approaches unavoidable. Further, changes in the atomic structure of material surfaces that can lead to surface reconstruction, and in the structure of proteins that can result in complete denaturation of the adsorbed molecules, can create many intermediate structural and energetic states that complicate sampling. In this review, we address the challenges posed to theoretical and computational methods in achieving accurate descriptions of the physical, chemical and mechanical properties of protein-surface systems. In this context, we discuss the applicability of different modeling and simulation techniques ranging from quantum mechanics through all-atom molecular mechanics to coarse-grained approaches. We examine uses of different sampling methods, as well as free energy calculations. Furthermore, we review computational studies of protein-surface interactions and discuss the successes and limitations of current approaches.

Background: Cytochrome P450 sterol 14α-demethylase (CYP51) is an essential enzyme for sterol biosynthesis and a target for anti-parasitic drug design. However, the design of parasite-specific drugs that inhibit parasitic CYP51 without severe side effects remains challenging. The active site of CYP51 is situated in the interior of the protein. Here, we characterize the potential ligand egress routes and mechanisms in Trypanosoma brucei and human CYP51 enzymes., Methods: We performed Random Acceleration Molecular Dynamics simulations of the egress of four different ligands from the active site of models of soluble and membrane-bound T. brucei CYP51 and of soluble human CYP51., Results: In the simulations, tunnel 2f, which leads to the membrane, was found to be the predominant ligand egress tunnel for all the ligands studied. Tunnels S, 1 and W, which lead to the cytosol, were also used in T. brucei CYP51, whereas tunnel 1 was the only other tunnel used significantly in human CYP51. The common tunnels found previously in other CYPs were barely used. The ligand egress times were shorter for human than T. brucei CYP51, suggesting lower barriers to ligand passage. Two gating residues, F105 and M460, in T. brucei CYP51 that modulate the opening of tunnels 2f and S were identified., Conclusions: Although the main egress tunnel was the same, differences in the tunnel-lining residues, ligand passage and tunnel usage were found between T. brucei and human CYP51s., General Significance: The results provide a basis for the design of selective anti-parasitic agents targeting the ligand tunnels., (Copyright © 2015 Elsevier B.V. All rights reserved.)

An important step in the simulation of a membrane protein in a phospholipid bilayer is the correct immersion of the protein in the bilayer. Crystal structures are determined without the bilayer. Particularly for proteins with monotopic domains, it can be unclear how deeply and in which orientation the protein is being inserted in the membrane. We have previously developed a procedure combining coarse-grain (CG) with all-atom (AA) molecular dynamics (MD) simulations to insert and simulate a cytochrome P450 (CYP) possessing an N-terminal transmembrane helix connected by a flexible linker region to a globular domain that dips into the membrane. The CG simulations provide a computationally efficient means to explore different orientations and conformations of the CYP in the membrane. Converged configurations obtained in the CG simulations are then refined in AA simulations. Here, we tested different variants of the MARTINI CG model, differing in the water model, the treatment of long-range non-bonded interactions, and the implementation (GROMACS 4.5.5 vs 5.0.4), for this purpose. We examined the behavior of the models for simulating a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer in water and for the immersion of CYP3A4 in a POPC bilayer, and compared the CG-MD results with the previously reported experimental and simulation results. We also tested the methodology on a set of four other CYPs. Finally, we propose an optimized protocol for modeling such protein-membrane systems that provides the most plausible configurations and is computationally efficient; this incorporates the standard non-polar water model and the GROMACS 5.0.4 implementation with a reaction field treatment of long-range interactions.

Conservation of function across families of orthologous enzymes is generally accompanied by conservation of their active site electrostatic potentials. To study the electrostatic conservation in the highly conserved essential enzyme, thymidylate synthase (TS), we conducted a systematic species-based comparison of the electrostatic potential in the vicinity of its active site. Whereas the electrostatics of the active site of TS are generally well conserved, the TSs from minimal organisms do not conform to the overall trend. Since the genomes of minimal organisms have a high thymidine content compared to other organisms, the observation of non-conserved electrostatics was surprising. Analysis of the symbiotic relationship between minimal organisms and their hosts, and the genetic completeness of the thymidine synthesis pathway suggested that TS from the minimal organism Wigglesworthia glossinidia (W.g.b.) must be active. Four residues in the vicinity of the active site of Escherichia coli TS were mutated individually and simultaneously to mimic the electrostatics of W.g.b TS. The measured activities of the E. coli TS mutants imply that conservation of electrostatics in the region of the active site is important for the activity of TS, and suggest that the W.g.b. TS has the minimal activity necessary to support replication of its reduced genome.

Fluorescent labels are often attached to proteins to monitor binding and adsorption processes. Docking simulations for native hen egg white lysozyme (HEWL) and HEWL labeled with fluorescein isothiocyanate show that these adsorb differently on charged surfaces. Attachment of even a small label can significantly change the interaction properties of a protein. Thus, the results of experiments with fluorescently labeled proteins should be interpreted by modeling the structures and computing the interaction properties of both labeled and unlabeled species.

The experimentally determined structures of human dipeptidyl peptidase III (DPP III) for the wild-type protein and for the complex of its E451A mutant with the peptide substrate, tynorphin, differ significantly in their overall shape. The two domains of the enzyme are separated by a wide cleft in the structure of the ligand-free enzyme, while in the ligand-bound mutant they are very close to each other, and the protein structure is extremely compact. Here, we applied a range of molecular dynamics simulation techniques to investigate the DPP III conformational landscape and the influence of ligand binding on the protein structure and dynamics. We used conventional, accelerated and steered methods to simulate DPP III and its complexes with tynorphin and with the preferred, synthetic, substrate Arg-Arg-2-naphthylamide. We found that DPP III can adopt a number of different forms in solution. The compact forms are more stable, but the open and partially closed states, spanning a wide range of conformations, can more effectively recognize the substrate which preferentially binds to the five-stranded β-core of the lower DPP III domain. The simulations indicated the existence of a dynamic equilibrium between open and semi-closed states and revealed two ways that the protein can close, leading to two distinct compact structures. The way in which the protein closes depends on the presence of the ligand.

The conformational properties of unbound multi-Cys2 His2 (mC2H2) zinc finger proteins, in which zinc finger domains are connected by flexible linkers, are studied by a multiscale approach. Three methods on different length scales are utilized. First, atomic detail molecular dynamics simulations of one zinc finger and its adjacent flexible linker confirmed that the zinc finger is more rigid than the flexible linker. Second, the end-to-end distance distributions of mC2H2 zinc finger proteins are computed using an efficient atomistic pivoting algorithm, which only takes excluded volume interactions into consideration. The end-to-end distance distribution gradually changes its profile, from left-tailed to right-tailed, as the number of zinc fingers increases. This is explained by using a worm-like chain model. For proteins of a few zinc fingers, an effective bending constraint favors an extended conformation. Only for proteins containing more than nine zinc fingers, is a somewhat compacted conformation preferred. Third, a mesoscale model is modified to study both the local and the global conformational properties of multi-C2H2 zinc finger proteins. Simulations of the CCCTC-binding factor (CTCF), an important mC2H2 zinc finger protein for genome spatial organization, are presented., (© 2015 Wiley Periodicals, Inc.)

Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states. Healthy metazoan cells effectively eliminate intracellular protein aggregates, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control.

The simulation of diffusional association (SDA) Brownian dynamics software package has been widely used in the study of biomacromolecular association. Initially developed to calculate bimolecular protein-protein association rate constants, it has since been extended to study electron transfer rates, to predict the structures of biomacromolecular complexes, to investigate the adsorption of proteins to inorganic surfaces, and to simulate the dynamics of large systems containing many biomacromolecular solutes, allowing the study of concentration-dependent effects. These extensions have led to a number of divergent versions of the software. In this article, we report the development of the latest version of the software (SDA 7). This release was developed to consolidate the existing codes into a single framework, while improving the parallelization of the code to better exploit modern multicore shared memory computer architectures. It is built using a modular object-oriented programming scheme, to allow for easy maintenance and extension of the software, and includes new features, such as adding flexible solute representations. We discuss a number of application examples, which describe some of the methods available in the release, and provide benchmarking data to demonstrate the parallel performance., (© 2015 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc.)

Macromolecular interactions play a crucial role in biological systems. Simulation of diffusional association (SDA) is a software for carrying out Brownian dynamics simulations that can be used to study the interactions between two or more biological macromolecules. webSDA allows users to run Brownian dynamics simulations with SDA to study bimolecular association and encounter complex formation, to compute association rate constants, and to investigate macromolecular crowding using atomically detailed macromolecular structures. webSDA facilitates and automates the use of the SDA software, and offers user-friendly visualization of results. webSDA currently has three modules: 'SDA docking' to generate structures of the diffusional encounter complexes of two macromolecules, 'SDA association' to calculate bimolecular diffusional association rate constants, and 'SDA multiple molecules' to simulate the diffusive motion of hundreds of macromolecules. webSDA is freely available to all users and there is no login requirement. webSDA is available at http://mcm.h-its.org/webSDA/., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The yeast spindle pole body (SPB) is the functional equivalent of the mammalian centrosome. The half bridge is a SPB substructure on the nuclear envelope (NE), playing a key role in SPB duplication. Its cytoplasmic components are the membrane-anchored Kar1, the yeast centrin Cdc31, and the Cdc31-binding protein Sfi1. In G1, the half bridge expands into the bridge through Sfi1 C-terminal (Sfi1-CT) dimerization, the licensing step for SPB duplication. We exploited photo-activated localization microscopy (PALM) to show that Kar1 localizes in the bridge center. Binding assays revealed direct interaction between Kar1 and C-terminal Sfi1 fragments. kar1Δ cells whose viability was maintained by the dominant CDC31-16 showed an arched bridge, indicating Kar1's function in tethering Sfi1 to the NE. Cdc31-16 enhanced Cdc31-Cdc31 interactions between Sfi1-Cdc31 layers, as suggested by binding free energy calculations. In our model, Kar1 binding is restricted to Sfi1-CT and Sfi1 C-terminal centrin-binding repeats, and centrin and Kar1 provide cross-links, while Sfi1-CT stabilizes the bridge and ensures timely SPB separation., (© 2015 Seybold et al.)

Human thymidylate synthase (hTS), a target for antiproliferative drugs, is an obligate homodimer. Single-point mutations to alanine at the monomer-monomer interface may enable the identification of specific residues that delineate sites for drugs aimed at perturbing the protein-protein interactions critical for activity. We computationally identified putative hotspot residues at the interface and designed mutants to perturb the intersubunit interaction. Dimer dissociation constants measured by a FRET-based assay range from 60 nM for wild-type hTS up to about 1 mM for single-point mutants and agree with computational predictions of the effects of these mutations. Mutations that are remote from the active site retain full or partial activity, although the substrate KM values were generally higher and the dimer was less stable. The lower dimer stability of the mutants can facilitate access to the dimer interface by small molecules and thereby aid the design of inhibitors that bind at the dimer interface.

Unlabelled: LigDig is a web server designed to answer questions that previously required several independent queries to diverse data sources. It also performs basic manipulations and analyses of the structures of protein-ligand complexes. The LigDig webserver is modular in design and consists of seven tools, which can be used separately, or via linking the output from one tool to the next, in order to answer more complex questions. Currently, the tools allow a user to: (i) perform a free-text compound search, (ii) search for suitable ligands, particularly inhibitors, of a protein and query their interaction network, (iii) search for the likely function of a ligand, (iv) perform a batch search for compound identifiers, (v) find structures of protein-ligand complexes, (vi) compare three-dimensional structures of ligand binding sites and (vii) prepare coordinate files of protein-ligand complexes for further calculations., Availability and Implementation: LigDig makes use of freely available databases, including ChEMBL, PubChem and SABIO-RK, and software programs, including cytoscape.js, PDB2PQR, ProBiS and Fconv. LigDig can be used by non-experts in bio- and chemoinformatics. LigDig is available at: http://mcm.h-its.org/ligdig., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2014. Published by Oxford University Press.)

Sterol 14α-demethylase (cytochrome P450 family 51 (CYP51)) is an essential enzyme occurring in all biological kingdoms. In eukaryotes, it is located in the membrane of the endoplasmic reticulum. Selective inhibitors of trypanosomal CYP51s that do not affect the human CYP51 have been discovered in vitro and found to cure acute and chronic mouse Chagas disease without severe side effects in vivo. Crystal structures indicate that CYP51 may be more rigid than most CYPs, and it has been proposed that this property may facilitate antiparasitic drug design. Therefore, to investigate the dynamics of trypanosomal CYP51, we built a model of membrane-bound Trypanosoma brucei CYP51 and then performed molecular dynamics simulations of T. brucei CYP51 in membrane-bound and soluble forms. We compared the dynamics of T. brucei CYP51 with those of human CYP51, CYP2C9, and CYP2E1. In the simulations, the CYP51s display low mobility in the buried active site although overall mobility is similar in all the CYPs studied. The simulations suggest that in CYP51, pathway 2f serves as the major ligand access tunnel, and both pathways 2f (leading to membrane) and S (leading to solvent) can serve as ligand egress tunnels. Compared with the other CYPs, the residues at the entrance of the ligand access tunnels in CYP51 have higher mobility that may be necessary to facilitate the passage of its large sterol ligands. The water (W) tunnel is accessible to solvent during most of the simulations of CYP51, but its width is affected by the conformations of the heme's two propionate groups. These differ from those observed in the other CYPs studied because of differences in their hydrogen-bonding network. Our simulations give insights into the dynamics of CYP51 that complement the available experimental data and have implications for drug design against CYP51 enzymes., (Copyright © 2015 John Wiley & Sons, Ltd.)

Many signaling events require the binding of cytoplasmic proteins to cell membranes by recognition of specific charged lipids, such as phosphoinositol-phosphates. As a model for a protein-membrane binding site, we consider one charged phosphoinositol phosphate (PtdIns(3)P) embedded in a phosphatidylcholine bilayer. As the protein-membrane binding is driven by electrostatic interactions, continuum solvent models require an accurate representation of the electrostatic potential of the phosphoinositol phosphate-containing membrane. We computed and analyzed the electrostatic potentials of snapshots taken at regular intervals from molecular dynamics simulations of the bilayer. We observe considerable variation in the electrostatic potential of the bilayer both along a single simulation and between simulations performed with the GAFF or CHARMM c36 force fields. However, we find that the choice of GAFF or CHARMM c36 parameters has little effect on the electrostatic potential of a given configuration of the bilayer with a PtdIns(3)P embedded in it. From our results, we propose a remedian averaging method for calculating the electrostatic potential of a membrane system that is suitable for simulations of protein-membrane binding with a continuum solvent model.

Background: Brownian dynamics (BD) simulations can be used to study very large molecular systems, such as models of the intracellular environment, using atomic-detail structures. Such simulations require strategies to contain the computational costs, especially for the computation of interaction forces and energies. A common approach is to compute interaction forces between macromolecules by precomputing their interaction potentials on three-dimensional discretized grids. For long-range interactions, such as electrostatics, grid-based methods are subject to finite size errors. We describe here the implementation of a Debye-Hückel correction to the grid-based electrostatic potential used in the SDA BD simulation software that was applied to simulate solutions of bovine serum albumin and of hen egg white lysozyme., Results: We found that the inclusion of the long-range electrostatic correction increased the accuracy of both the protein-protein interaction profiles and the protein diffusion coefficients at low ionic strength., Conclusions: An advantage of this method is the low additional computational cost required to treat long-range electrostatic interactions in large biomacromolecular systems. Moreover, the implementation described here for BD simulations of protein solutions can also be applied in implicit solvent molecular dynamics simulations that make use of gridded interaction potentials.

Both subunits of αβ-tubulin that comprise the core components of microtubules bind GTP. GTP binding to α-tubulin has a structural role, whereas β-tubulin binds and hydrolyses GTP to regulate microtubule dynamics. γ-tubulin, another member of the tubulin superfamily that seeds microtubule nucleation at microtubule-organizing centres, also binds GTP; however, the importance of this association remains elusive. To address the role of GTP binding to γ-tubulin, we systematically mutagenized the GTP contact residues in the yeast γ-tubulin Tub4. Tub4(GTP)-mutant proteins that exhibited greatly reduced GTP affinity still assembled into the small γ-tubulin complex. However, tub4(GTP) mutants were no longer viable, and had defects in interaction between γ-tubulin and αβ-tubulin, decreased microtubule nucleation and defects in microtubule organization. In vitro and in vivo data show that only γ-tubulin loaded with GTP nucleates microtubules. Our results suggest that GTP recruitment to γ-tubulin enhances its interaction with αβ-tubulin similarly to GTP recruitment to β-tubulin.

Despite high similarity in sequence and catalytic properties, the l-lactate dehydrogenases (LDHs) in lactic acid bacteria (LAB) display differences in their regulation that may arise from their adaptation to different habitats. We combined experimental and computational approaches to investigate the effects of fructose 1,6-bisphosphate (FBP), phosphate (Pi), and ionic strength (NaCl concentration) on six LDHs from four LABs studied at pH 6 and pH 7. We found that 1) the extent of activation by FBP (Kact) differs. Lactobacillus plantarum LDH is not regulated by FBP, but the other LDHs are activated with increasing sensitivity in the following order: Enterococcus faecalis LDH2 ≤ Lactococcus lactis LDH2 < E. faecalis LDH1 < L. lactis LDH1 ≤ Streptococcus pyogenes LDH. This trend reflects the electrostatic properties in the allosteric binding site of the LDH enzymes. 2) For L. plantarum, S. pyogenes, and E. faecalis, the effects of Pi are distinguishable from the effect of changing ionic strength by adding NaCl. 3) Addition of Pi inhibits E. faecalis LDH2, whereas in the absence of FBP, Pi is an activator of S. pyogenes LDH, E. faecalis LDH1, and L. lactis LDH1 and LDH2 at pH 6. These effects can be interpreted by considering the computed binding affinities of Pi to the catalytic and allosteric binding sites of the enzymes modeled in protonation states corresponding to pH 6 and pH 7. Overall, the results show a subtle interplay among the effects of Pi, FBP, and pH that results in different regulatory effects on the LDHs of different LABs.

We present TRAPP (TRAnsient Pockets in Proteins), a new automated software platform for tracking, analysis, and visualization of binding pocket variations along a protein motion trajectory or within an ensemble of protein structures that may encompass conformational changes ranging from local side chain fluctuations to global backbone motions. TRAPP performs accurate grid-based calculations of the shape and physicochemical characteristics of a binding pocket for each structure and detects the conserved and transient regions of the pocket in an ensemble of protein conformations. It also provides tools for tracing the opening of a particular subpocket and residues that contribute to the binding site. TRAPP thus enables an assessment of the druggability of a disease-related target protein taking its flexibility into account.

As a model for understanding how molecular crowding influences diffusion and transport of proteins in cellular environments, we combined experimental and theoretical approaches to study the diffusion of proteins in highly concentrated protein solutions. Bovine serum albumin and γ-Globulin were chosen as molecular crowders and as tracers. These two proteins are representatives of the main types of plasma protein and have different shapes and sizes. Solutions consisting of one or both proteins were studied. The self-diffusion coefficients of the fluorescently labeled tracer proteins were measured by means of fluorescence correlation spectroscopy at a total protein concentration of up to 400 g/L. γ-Globulin is found to have a stronger influence as a crowder on the tracer self-diffusion coefficient than Bovine serum albumin. Brownian dynamics simulations show that the excluded volume and the shape of the crowding protein have a significantly stronger influence on translational and rotational diffusion coefficients, as well as transient oligomerization, than hydrodynamic or direct interactions. Anomalous subdiffusion, which is not observed at the experimental fluorescence correlation spectroscopy timescales (>100 μs), appears only at very short timescales (<1 μs) in the simulations due to steric effects of the proteins. We envision that the combined experimental and computational approach employed here can be developed to unravel the different biophysical contributions to protein motion and interaction in cellular environments by systematically varying protein properties such as molecular weight, size, shape, and electrostatic interactions., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Resistance to drugs targeting human thymidylate synthase (TS) poses a major challenge in the field of anti-cancer therapeutics. Overexpression of the TS protein has been implicated as one of the factors leading to the development of resistance. Therefore, repressing translation by targeting the TS mRNA could help to overcome this problem. In this study, we report that the compound Hoechst 33258 (HT) can reduce cellular TS protein levels without altering TS mRNA levels, suggesting that it modulates TS expression at the translation level. We have combined nuclear magnetic resonance, UV-visible and fluorescence spectroscopy methods with docking and molecular dynamics simulations to study the interaction of HT with a region in the TS mRNA. The interaction predominantly involves intercalation of HT at a CC mismatch in the region near the translational initiation site. Our results support the use of HT-like compounds to guide the design of therapeutic agents targeting TS mRNA.

Phenolnaphthalein derivatives show potential for pharmacological activity as inhibitors of thymidylate synthase (TS) but difficulties in their synthesis and derivatization hinder their development. A deconstruction approach aimed at identifying a suitable new scaffold was proposed. A new scaffold was identified and two compound libraries based on this scaffold were designed. The carboxamide library (Library B) showed specific inhibition activity against Escherichia coli TS, whereas the sulfonamide library (Library C) showed a non-specific inhibition profile against hTS. N-(1,3-Dioxo-1H,3H-benzo[de]isochromen-5-yl)-sulfonamide derivatives, 1C and 9C, showed one order of magnitude improvement in inhibition constant against hTS with respect to the starting lead and represent potential compounds for further lead development., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The mammalian cytochrome P450 (CYP) enzymes play important roles in drug metabolism, steroid biosynthesis, and xenobiotic degradation. The active site of CYPs is buried in the protein and thus the ligands have to enter and exit the active site via ligand tunnels. Conformational changes of flexible parts of the protein usually accompany the entrance and exit of ligands. Comparison of the crystal structures of mammalian CYPs in closed, open, and partially open states reveals that the greatest conformational diversity associated with ligand tunnel opening is in the regions of the B-C and F-G loops. Some CYPs have been observed to adopt different open and closed conformations when bound to different ligands, suggesting that the ligand entrance and exit routes might differ according to the ligand properties. Mammalian CYPs are mostly membrane-bound enzymes, making them difficult to characterize structurally and dynamically. A range of molecular dynamics simulation techniques has been applied to investigate the dynamics and the ligand tunnels of these proteins both in the aqueous environment, and more recently, in lipid bilayers. These simulations not only reveal multiple tunnels through which ligands can pass but also show that different tunnels are preferred by different ligands and that the lipid bilayer can influence the protein dynamics and tunnel opening. The results indicate that not only the active site but also the ligand tunnels can contribute to the different substrate specificity profiles of the mammalian CYPs., (© 2013 International Union of Biochemistry and Molecular Biology, Inc.)

Pyruvate kinase (PYK) is a critical allosterically regulated enzyme that links glycolysis, the primary energy metabolism, to cellular metabolism. Lactic acid bacteria rely almost exclusively on glycolysis for their energy production under anaerobic conditions, which reinforces the key role of PYK in their metabolism. These organisms are closely related, but have adapted to a huge variety of native environments. They include food-fermenting organisms, important symbionts in the human gut, and antibiotic-resistant pathogens. In contrast to the rather conserved inhibition of PYK by inorganic phosphate, the activation of PYK shows high variability in the type of activating compound between different lactic acid bacteria. System-wide comparative studies of the metabolism of lactic acid bacteria are required to understand the reasons for the diversity of these closely related microorganisms. These require knowledge of the identities of the enzyme modifiers. Here, we predict potential allosteric activators of PYKs from three lactic acid bacteria which are adapted to different native environments. We used protein structure-based molecular modeling and enzyme kinetic modeling to predict and validate potential activators of PYK. Specifically, we compared the electrostatic potential and the binding of phosphate moieties at the allosteric binding sites, and predicted potential allosteric activators by docking. We then made a kinetic model of Lactococcus lactis PYK to relate the activator predictions to the intracellular sugar-phosphate conditions in lactic acid bacteria. This strategy enabled us to predict fructose 1,6-bisphosphate as the sole activator of the Enterococcus faecalis PYK, and to predict that the PYKs from Streptococcus pyogenes and Lactobacillus plantarum show weaker specificity for their allosteric activators, while still having fructose 1,6-bisphosphate play the main activator role in vivo. These differences in the specificity of allosteric activation may reflect adaptation to different environments with different concentrations of activating compounds. The combined computational approach employed can readily be applied to other enzymes.

The ring-forming AAA+ protein ClpB cooperates with the DnaK chaperone system to refold aggregated proteins in Escherichia coli. The M domain, a ClpB-specific coiled-coil structure with two wings, motif 1 and motif 2, is essential to disaggregation, but the positioning and mechanistic role of M domains in ClpB hexamers remain unresolved. We show that M domains nestle at the ClpB ring surface, with both M-domain motifs contacting the first ATPase domain (AAA-1). Both wings contribute to maintaining a repressed ClpB activity state. Motif 2 docks intramolecularly to AAA-1 to regulate ClpB unfolding power, and motif 1 contacts a neighboring AAA-1 domain. Mutations that stabilize motif 2 docking repress ClpB, whereas destabilization leads to derepressed ClpB activity with greater unfolding power that is toxic in vivo. Our results underline the vital nature of tight ClpB activity control and elucidate a regulated M-domain toggle control mechanism.

Protein-nanoparticle associations have important applications in nanoscience and nanotechnology such as targeted drug delivery and theranostics. However, the mechanisms by which proteins recognize nanoparticles and the determinants of specificity are still poorly understood at the microscopic level. Gold is a promising material in nanoparticles for nanobiotechnology applications because of the ease of its functionalization and its tunable optical properties. Ubiquitin is a small, cysteine-free protein (ubiquitous in eukaryotes) whose binding to gold nanoparticles has been characterized recently by nuclear magnetic resonance (NMR). To reveal the molecular basis of these protein-nanoparticle interactions, we performed simulations at multiple levels (ab initio quantum mechanics, classical molecular dynamics and Brownian dynamics) and compared the results with experimental data (circular dichroism and NMR). The results provide a model of the ensemble of structures constituting the ubiquitin-gold surface complex, and insights into the driving forces for the binding of ubiquitin to gold nanoparticles, the role of nanoparticle surfactants (citrate) in the association process, and the origin of the perturbations in the NMR chemical shifts.

Many enzymes have buried active sites. The properties of the tunnels connecting the active site with bulk solvent affect ligand binding and unbinding and also the catalytic properties. Here, we investigate ligand passage in the haloalkane dehalogenase enzyme LinB and the effect of replacing leucine by a bulky tryptophan at a tunnel-lining position. Transient kinetic experiments show that the mutation significantly slows down the rate of product release. Moreover, the mechanism of bromide ion release is changed from a one-step process in the wild type enzyme to a two-step process in the mutant. The rate constant of bromide ion release corresponds to the overall steady-state turnover rate constant, suggesting that product release became the rate-limiting step of catalysis in the mutant. We explain the experimental findings by investigating the molecular details of the process computationally. Analysis of trajectories from molecular dynamics simulations with a tunnel detection software reveals differences in the tunnels available for ligand egress. Corresponding differences are seen in simulations of product egress using a specialized enhanced sampling technique. The differences in the free energy barriers for egress of a bromide ion obtained using potential of mean force calculations are in good agreement with the differences in rates obtained from the transient kinetic experiments. Interactions of the bromide ion with the introduced tryptophan are shown to affect the free energy barrier for its passage. The study demonstrates how the mechanism of an enzymatic catalytic cycle and reaction kinetics can be engineered by modification of protein tunnels.

High macromolecular concentrations are a distinguishing feature of living organisms. Understanding how the high concentration of solutes affects the dynamic properties of biological macromolecules is fundamental for the comprehension of biological processes in living systems. In this paper, we describe the implementation of mean field models of translational and rotational hydrodynamic interactions into an atomically detailed many-protein brownian dynamics simulation method. Concentrated solutions (30-40% volume fraction) of myoglobin, hemoglobin A, and sickle cell hemoglobin S were simulated, and static structure factors, oligomer formation, and translational and rotational self-diffusion coefficients were computed. Good agreement of computed properties with available experimental data was obtained. The results show the importance of both solvent mediated interactions and weak protein-protein interactions for accurately describing the dynamics and the association properties of concentrated protein solutions. Specifically, they show a qualitative difference in the translational and rotational dynamics of the systems studied. Although the translational diffusion coefficient is controlled by macromolecular shape and hydrodynamic interactions, the rotational diffusion coefficient is affected by macromolecular shape, direct intermolecular interactions, and both translational and rotational hydrodynamic interactions.

Plants and bacteria assimilate sulfur into cysteine. Cysteine biosynthesis involves a bienzyme complex, the cysteine synthase complex (CSC), which consists of serine-acetyl-transferase (SAT) and O-acetyl-serine-(thiol)-lyase (OAS-TL) enzymes. The activity of OAS-TL is reduced by formation of the CSC. Although this reduction is an inherent part of the self-regulation cycle of cysteine biosynthesis, there has until now been no explanation as to how OAS-TL loses activity in plants. Complexation of SAT and OAS-TL involves binding of the C-terminal tail of SAT in one of the active sites of the homodimeric OAS-TL. We here explore the flexibility of the unoccupied active site in Arabidopsis thaliana cytosolic and mitochondrial OAS-TLs. Our results reveal two gates in the OAS-TL active site that define its accessibility. The observed dynamics of the gates show allosteric closure of the unoccupied active site of OAS-TL in the CSC, which can hinder substrate binding, abolishing its turnover to cysteine., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The active site of liver-specific, drug-metabolizing cytochrome P450 (CYP) monooxygenases is deeply buried in the protein and is connected to the protein surface through multiple tunnels, many of which were found open in different CYP crystal structures. It has been shown that different tunnels could serve as ligand passage routes in different CYPs. However, it is not understood whether one CYP uses multiple routes for substrate access and product release and whether these routes depend on ligand properties. From 300 ns of molecular dynamics simulations of CYP2C9, the second most abundant CYP in the human liver we found four main ligand exit routes, the occurrence of each depending on the ligand type and the conformation of the F-G loop, which is likely to be affected by the CYP-membrane interaction. A non-helical F-G loop favored exit towards the putative membrane-embedded region. Important protein features that direct ligand exit include aromatic residues that divide the active site and whose motions control access to two pathways. The ligands interacted with positively charged residues on the protein surface through hydrogen bonds that appear to select for acidic substrates. The observation of multiple, ligand-dependent routes in a CYP aids understanding of how CYP mutations affect drug metabolism and provides new possibilities for CYP inhibition.