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Recent advances in computational protein design now enable the massively parallel de novo design and experimental characterization of small hyperstable binding proteins with potential therapeutic activity. By providing experimental feedback on tens of thousands of designed proteins, the design‐build‐test‐learn pipeline provides a unique opportunity to systematically improve our understanding of protein folding and binding. Here, we review the structures of mini‐protein binders in complex with Influenza hemagglutinin and Bot toxin, and illustrate in the case of disulfide bond placement how analysis of the large datasets of computational models and experimental data can be used to identify determinants of folding and binding. [ABSTRACT FROM AUTHOR]

The RNA polymerase II (Pol II) is a eukaryotic enzyme that catalyzes the synthesis of the messenger RNA using a DNA template. Despite numerous biochemical and biophysical studies, it remains elusive whether the “secondary channel” is the only route for NTP to reach the active site of the enzyme or if the “main channel” could be an alternative. On this regard, crystallographic structures of Pol II have been extremely useful to understand the structural basis of transcription, however, the conformation of the unpaired non-template DNA part of the full transcription bubble (TB) is still unknown. Since diffusion routes of the nucleoside triphosphate (NTP) substrate through the main channel might overlap with the TB region, gaining structural information of the full TB is critical for a complete understanding of Pol II transcription process. In this study, we have built a structural model of Pol II with a complete transcription bubble based on multiple sources of existing structural data and used Molecular Dynamics (MD) simulations together with structural analysis to shed light on NTP entry pathways. Interestingly, we found that although both channels have enough space to allow NTP loading, the percentage of MD conformations containing enough space for NTP loading through the secondary channel is twice higher than that of the main channel. Further energetic study based on MD simulations with NTP loaded in the channels has revealed that the diffusion of the NTP through the main channel is greatly disfavored by electrostatic repulsion between the NTP and the highly negatively charged backbones of nucleotides in the non-template DNA strand. Taken together, our results suggest that the secondary channel is the major route for NTP entry during Pol II transcription. [ABSTRACT FROM AUTHOR]

The rational designing of binding abilities in proteins requires an understanding of the relationship between structure and thermodynamics. However, our knowledge of the molecular origin of high-affinity binding of ligands to proteins is still limited; such is the case for l-lysine- l-arginine- l-ornithine periplasmic binding protein (LAOBP), a periplasmic binding protein from Salmonella typhimurium that binds to l-arginine, l-lysine, and l-ornithine with nanomolar affinity and to l-histidine with micromolar affinity. Structural studies indicate that ligand binding induces a large conformational change in LAOBP. In this work, we studied the thermodynamics of l-histidine and l-arginine binding to LAOBP by isothermal titration calorimetry. For both ligands, the affinity is enthalpically driven, with a binding Δ Cp of ~−300 cal mol−1 K−1, most of which arises from the burial of protein nonpolar surfaces that accompanies the conformational change. Osmotic stress measurements revealed that several water molecules become sequestered upon complex formation. In addition, LAOBP prefers positively charged ligands in their side chain. An energetic analysis shows that the protein acquires a thermodynamically equivalent state with both ligands. The 1000-fold higher affinity of LAOBP for l-arginine as compared with l-histidine is mainly of enthalpic origin and can be ascribed to the formation of an extra pair of hydrogen bonds. Periplasmic binding proteins have evolved diverse energetic strategies for ligand recognition. STM4351, another arginine binding protein from Salmonella, shows an entropy-driven micromolar affinity toward l-arginine. In contrast, our data show that LAOBP achieves nanomolar affinity for the same ligand through enthalpy optimization. Copyright © 2015 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Protein-ligand recognition plays key roles in many biological processes. One of the most fascinating questions about protein-ligand recognition is to understand its underlying mechanism, which often results from a combination of induced fit and conformational selection. In this study, we have developed a three-pronged approach of Markov State Models, Molecular Dynamics simulations, and flux analysis to determine the contribution of each model. Using this approach, we have quantified the recognition mechanism of the choline binding protein (ChoX) to be ∼90% conformational selection dominant under experimental conditions. This is achieved by recovering all the necessary parameters for the flux analysis in combination with available experimental data. Our results also suggest that ChoX has several metastable conformational states, of which an apo-closed state is dominant, consistent with previous experimental findings. Our methodology holds great potential to be widely applied to understand recognition mechanisms underlining many fundamental biological processes. [ABSTRACT FROM AUTHOR]

Transcription is a central step in gene expression, in which the DNA template is processively read by RNA polymerase II (Pol II), synthesizing a complementary messenger RNA transcript. At each cycle, Pol II moves exactly one register along the DNA, a process known as translocation. Although X-ray crystal structures have greatly enhanced our understanding of the transcription process, the underlying molecular mechanisms of translocation remain unclear. Here we use sophisticated simulation techniques to observe Pol II translocation on a millisecond timescale and at atomistic resolution. We observe multiple cycles of forward and backward translocation and identify two previously unidentified intermediate states. We show that the bridge helix (BH) plays a key role accelerating the translocation of both the RNA:DNA hybrid and transition nucleotide by directly interacting with them. The conserved BH residues, Thr831 and Tyr836, mediate these interactions. To date, this study delivers the most detailed picture of the mechanism of Pol II translocation at atomic level. [ABSTRACT FROM AUTHOR]

We present a set of force field (FF) parameters compatible with the AMBER03 FF to describe five cofactors in photosystem II (PSII) of oxygenic photosynthetic organisms: plastoquinone-9 (three redox forms), chlorophyll-a, pheophytin-a, heme-b, and β-carotene. The development of a reliable FF for these cofactors is an essential step for performing molecular dynamics simulations of PSII. Such simulations are important for the calculation of absorption spectrum and the further investigation of the electron and energy transfer processes. We have derived parameters for partial charges, bonds, angles, and dihedral-angles from solid theoretical models using systematic quantum mechanics (QM) calculations. We have shown that the developed FF parameters are in good agreement with both ab initio QM and experimental structural data in small molecule crystals as well as protein complexes. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Molecular recognition is determined by the structure and dynamics of both a protein and its ligand, but it is difficult to directly assess the role of each of these players. In this study, we use Markov State Models (MSMs) built from atomistic simulations to elucidate the mechanism by which the Lysine-, Arginine-, Ornithine-binding (LAO) protein binds to its ligand. We show that our model can predict the bound state, binding free energy, and association rate with reasonable accuracy and then use the model to dissect the binding mechanism. In the past, this binding event has often been assumed to occur via an induced fit mechanism because the protein's binding site is completely closed in the bound state, making it impossible for the ligand to enter the binding site after the protein has adopted the closed conformation. More complex mechanisms have also been hypothesized, but these have remained controversial. Here, we are able to directly observe roles for both the conformational selection and induced fit mechanisms in LAO binding. First, the LAO protein tends to form a partially closed encounter complex via conformational selection (that is, the apo protein can sample this state), though the induced fit mechanism can also play a role here. Then, interactions with the ligand can induce a transition to the bound state. Based on these results, we propose that MSMs built from atomistic simulations may be a powerful way of dissecting ligand-binding mechanisms and may eventually facilitate a deeper understanding of allostery as well as the prediction of new protein-ligand interactions, an important step in drug discovery. [ABSTRACT FROM AUTHOR]

Abstract: Chemokines are key molecules involved in the control of leukocyte trafficking. Recently, a novel function as antimicrobial proteins has been described. CCL13 is the only member of the MCP chemokine subfamily displaying antimicrobial activity. To determine the key residues involved in its antimicrobial activity, CCL13 derived peptides were synthesized and tested against several bacterial strains, including Pseudomonas aeruginosa. One of these peptides, corresponding to the C-terminal region of CCL13 (CDAP-4) displayed good antimicrobial activity. Electron microscopy studies revealed remarkable morphological changes after CDAP-4 treatment. By computer modeling, CDAP-4 in α helical configuration generated a positive electrostatic potential that extended beyond the surface of the molecule. This feature is similar to other antimicrobial peptides. Altogether, these findings indicate that the antimicrobial activity was displayed by CCL13 resides to some extent at the C-terminal region. Furthermore, CDAP-4 could be considered a good antimicrobial candidate with a potential use against pathogens including P. aeruginosa. [Copyright &y& Elsevier]

Substrate‐binding proteins (SBPs) are used by organisms from the three domains of life for transport and signalling. SBPs are composed of two domains that collectively trap ligands with high affinity and selectivity. To explore the role of the domains and the integrity of the hinge region between them in the function and conformation of SBPs, here, we describe the ligand binding, conformational stability and folding kinetics of the Lysine Arginine Ornithine (LAO) binding protein from Salmonella thiphimurium and constructs corresponding to its two independent domains. LAO is a class II SBP formed by a continuous and a discontinuous domain. Contrary to the expected behaviour based on their connectivity, the discontinuous domain shows a stable native‐like structure that binds l‐arginine with moderate affinity, whereas the continuous domain is barely stable and shows no detectable ligand binding. Regarding folding kinetics, studies of the entire protein revealed the presence of at least two intermediates. While the unfolding and refolding of the continuous domain exhibited only a single intermediate and simpler and faster kinetics than LAO, the folding mechanism of the discontinuous domain was complex and involved multiple intermediates. These findings suggest that in the complete protein the continuous domain nucleates folding and that its presence funnels the folding of the discontinuous domain avoiding nonproductive interactions. The strong dependence of the function, stability and folding pathway of the lobes on their covalent association is most likely the result of the coevolution of both domains as a single unit. [ABSTRACT FROM AUTHOR]

The emergence of novel variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need to investigate alternative approaches to prevent infection and treat patients with coronavirus disease 2019. Here, we report the preclinical efficacy of NL-CVX1, a de novo decoy that blocks virus entry into cells by binding with nanomolar affinity and high specificity to the receptor-binding domain of the SARS-CoV-2 spike protein. Using a transgenic mouse model of SARS-CoV-2 infection, we showed that a single prophylactic intranasal dose of NL-CVX1 conferred complete protection from severe disease following SARS-CoV-2 infection. Multiple therapeutic administrations of NL-CVX1 also protected mice from succumbing to infection. Finally, we showed that infected mice treated with NL-CVX1 developed both anti-SARS-CoV-2 antibodies and memory T cells and were protected against reinfection a month after treatment. Overall, these observations suggest NL-CVX1 is a promising therapeutic candidate for preventing and treating severe SARS-CoV-2 infections. [ABSTRACT FROM AUTHOR]

The therapeutic potential of recombinant cytokines has been limited by the severe side effects of systemic administration. We describe a strategy to reduce the dose-limiting toxicities of monomeric cytokines by designing two components that require colocalization for activity and that can be independently targeted to restrict activity to cells expressing two surface markers. We demonstrate the approach with a previously designed mimetic of cytokines interleukin-2 and interleukin-15—Neoleukin-2/15 (Neo-2/15)—both for trans-activating immune cells surrounding targeted tumor cells and for cis-activating directly targeted immune cells. In trans-activation mode, tumor antigen targeting of the two components enhanced antitumor activity and attenuated toxicity compared with systemic treatment in syngeneic mouse melanoma models. In cis-activation mode, immune cell targeting of the two components selectively expanded CD8+ T cells in a syngeneic mouse melanoma model and promoted chimeric antigen receptor T cell activation in a lymphoma xenograft model, enhancing antitumor efficacy in both cases. IL-2 cytokine therapy is made safer by splitting a designed mimetic in two for tumor targeting. [ABSTRACT FROM AUTHOR]

The initiation of transcription by RNA polymerase II is a multistage process. X-ray crystal structures of transcription complexes containing short RNAs reveal three structural states: one with 2- and 3-nucleotide RNAs, in which only the 3'-end of the RNA is detectable; a second state with 4- and 5-nucleotide RNAs, with an RNA-DNA hybrid in a grossly distorted conformation; and a third state with RNAs of 6 nucleotides and longer, essentially the same as a stable elongating complex. The transition from the first to the second state correlates with a markedly reduced frequency of abortive initiation. The transition from the second to the third state correlates with partial "bubble collapse" and promoter escape. Polymerase structure is permissive for abortive initiation, thereby setting a lower limit on polymerase-promoter complex lifetime and allowing the dissociation of nonspecific complexes. Abortive initiation may be viewed as promoter proofreading, and the structural transitions as checkpoints for promoter control. [ABSTRACT FROM AUTHOR]

We proposed a computational protocol of simulating the T-jump peptide unfolding experiments and the related transient IR and two-dimensional IR (2DIR) spectra based on the Markov state model (MSM) and nonlinear exciton propagation (NEP) methods. MSMs partition the conformation space into a set of nonoverlapping metastable states, and we can calculate spectra signal for each of these states using the NEP method. Thus the overall spectroscopic observable for a given system is simply the sum of spectra of different metastable states weighted by their populations. We show that results from MSMs constructed from a large number of simulations have a much better agreement with the equilibrium experimental 2DIR spectra compared to that generated from straightforward MD simulations starting from the folded state. This indicates that a sufficient sampling of important relevant conformational states is critical for calculating the accurate spectroscopic observables. MSMs are also capable of simulating the unfolding relaxation dynamics upon the temperature jump. The agreement of the simulation using MSMs and NEP with the experiment not only provides a justification for our protocol, but also provides the physical insight of the underlying spectroscopic observables. The protocol we developed has the potential to be extended to simulate a wide range of fast triggering plus optical detection experiments for biomolecules. [ABSTRACT FROM AUTHOR]

Engineered proteins are revolutionizing immunotherapy, but advances are still needed to harness their full potential. Traditional protein engineering methods use naturally existing proteins as a starting point, and therefore, are intrinsically limited to small alterations of a protein's natural structure and function. Conversely, computational de novo protein design is free of such limitation, and can produce a virtually infinite number of novel protein sequences, folds, and functions. Recently, we used de novo protein engineering to create Neoleukin-2/15 (Neo-2/15), a protein mimetic of the function of both interleukin-2 (IL-2) and interleukin-15 (IL-15). To our knowledge, Neo-2/15 is the first de novo protein with immunotherapeutic activity, and in murine cancer models, it has demonstrated enhanced therapeutic potency and reduced toxicity compared to IL-2. De novo protein design is already showcasing its tremendous potential for driving the next wave of protein-based therapeutics that are explicitly engineered to treat disease. [ABSTRACT FROM AUTHOR]

Mixed-chirality peptide macrocycles such as cyclosporine are among the most potent therapeutics identified to date, but there is currently no way to systematically search the structural space spanned by such compounds. Natural proteins do not provide a useful guide: Peptide macrocycles lack regular secondary structures and hydrophobic cores, and can contain local structures not accessible with L-amino acids. Here, we enumerate the stable structures that can be adopted by macrocyclic peptides composed of L- and D-amino acids by near-exhaustive backbone sampling followed by sequence design and energy landscape calculations. We identify more than 200 designs predicted to fold into single stable structures, many times more than the number of currently available unbound peptide macrocycle structures. Nuclear magnetic resonance structures of 9 of 12 designed 7- to 10-residue macrocycles, and three 11- to 14-residue bicyclic designs, are close to the computational models. Our results provide a nearly complete coverage of the rich space of structures possible for short peptide macrocycles and vastly increase the available starting scaffolds for both rational drug design and library selection methods. [ABSTRACT FROM AUTHOR]

The folding of natural proteins typically relies on hydrophobic packing, metal binding, or disulfide bond formation in the protein core. Alternatively, a 3D structure can be defined by incorporating a multivalent cross-linking agent, and this approach has been successfully developed for the selection of bicyclic peptides from large random-sequence libraries. By contrast, there is no general method for the de novo computational design of multicross-linked proteins with predictable and well-defined folds, including ones not found in nature. Here we use Rosetta and Tertiary Motifs (TERMs) to design small proteins that fold around multivalent cross-linkers. The hydrophobic cross-linkers stabilize the fold by macrocyclic restraints, and they also form an integral part of a small apolar core. The designed CovCore proteins were prepared by chemical synthesis, and their structures were determined by solution NMR or X-ray crystallography. These mesosized proteins, lying between conventional proteins and small peptides, are easily accessible either through biosynthetic precursors or chemical synthesis. The unique tertiary structures and ease of synthesis of CovCore proteins indicate that they should provide versatile templates for developing inhibitors of protein-protein interactions. [ABSTRACT FROM AUTHOR]

Active sites and ligand-binding cavities in native proteins are often formed by curved β sheets, and the ability to control β-sheet curvature would allow design of binding proteins with cavities customized to specific ligands. Toward this end, we investigated the mechanisms controlling β-sheet curvature by studying the geometry of β sheets in naturally occurring protein structures and folding simulations. The principles emerging from this analysis were used to design, de novo, a series of proteins with curved β sheets topped with α helices. Nuclear magnetic resonance and crystal structures of the designs closely match the computational models, showing that β-sheet curvature can be controlled with atomic-level accuracy. Our approach enables the design of proteins with cavities and provides a route to custom design ligand-binding and catalytic sites. [ABSTRACT FROM AUTHOR]

Many cancers overexpress one or more of the six human pro-survival BCL2 family proteins to evade apoptosis. To determine which BCL2 protein or proteins block apoptosis in different cancers, we computationally designed three-helix bundle protein inhibitors specific for each BCL2 pro-survival protein. Following in vitro optimization, each inhibitor binds its target with high picomolar to low nanomolar affinity and at least 300-fold specificity. Expression of the designed inhibitors in human cancer cell lines revealed unique dependencies on BCL2 proteins for survival which could not be inferred from other BCL2 profiling methods. Our results show that designed inhibitors can be generated for each member of a closely-knit protein family to probe the importance of specific protein-protein interactions in complex biological processes. [ABSTRACT FROM AUTHOR]

Markov state models (MSMs) have become a popular approach for investigating the conformational dynamics of proteins and other biomolecules. MSMs are typically built from numerous molecular dynamics simulations by dividing the sampled configurations into a large number of microstates based on geometric criteria. The resulting microstate model can then be coarse-grained into a more understandable macrostate model by lumping together rapidly mixing microstates into larger, metastable aggregates. However, finite sampling often results in the creation of many poorly sampled microstates. During coarse-graining, these states are mistakenly identified as being kinetically important because transitions to/from them appear to be slow. In this paper, we propose a formalism based on an algebraic principle for matrix approximation, i.e., the Nyström method, to deal with such poorly sampled microstates. Our scheme builds a hierarchy of microstates from high to low populations and progressively applies spectral clustering on sets of microstates within each level of the hierarchy. It helps spectral clustering identify metastable aggregates with highly populated microstates rather than being distracted by lowly populated states. We demonstrate the ability of this algorithm to discover the major metastable states on two model systems, the alanine dipeptide and trpzip2 peptide. [ABSTRACT FROM AUTHOR]