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The biochemical journal / Reviews 479(3), 337 - 356 (2022). doi:10.1042/BCJ20210829, As the decline of malaria cases stalled over the last five years, novel targets in Plasmodium falciparum are necessary for the development of new drugs. Glycogen Synthase Kinase (PfGSK3) has been identified as a potential target, since its selective inhibitors were shown to disrupt the parasitès life cycle. In the uncanonical N-terminal region of the parasite enzyme, we identified several autophosphorylation sites and probed their role in activity regulation of PfGSK3. By combining molecular modeling with experimental small-angle X-ray scattering data, we show that increased PfGSK3 activity is promoted by conformational changes in the PfGSK3 N-terminus, triggered by N-terminal phosphorylation. Our work provides novel insights into the structure and regulation of the malarial PfGSK3., Published by Portland Pr., London [u.a.]

Graphical abstract, Highlights • SAXS is a unique tool to study MP systems in the native-like environment. • The lack of user-friendly software hinders SAXS data interpretation of MPs. • We present a novel plugin for the widely used molecular visualization system PyMol. • MPBuilder allows to build and refine models against SAXS data., Membrane proteins (MPs) are the target of numerous structural and functional studies in biological and medical/pharmaceutical sciences. Strategies for the high-throughput structural analysis of MPs and of their perturbations driven by ligands having potential therapeutic applications are uncommon, often requiring scaled up crystallization, electron microscopy, and nuclear magnetic resonance (NMR) efforts. Small-angle X-ray scattering (SAXS) provides a rapid means to study low resolution structures and conformational changes of native MPs in solution without cumbersome sample preparations/treatment. The method requires the MPs solubilized in an appropriate medium (eg. detergents, mixed micelles and nanodiscs) and reliable and robust models are needed to describe the relevant complexes. Here we present MPBuilder, a simple and versatile tool for the generation and refinement of all-atom MP systems in the popular software PyMOL, an environment familiar to most biologists. MPBuilder provides building capability for protein-detergent, bicelle, and lipid-scaffold (saposin nanoparticles, nanodiscs) complexes and links this to the ATSAS software package modules for model refinement and validation against the SAXS data.

Scientific reports 10(1), 8081 (2020). doi:10.1038/s41598-020-64933-1, The application of small angle X-ray scattering (SAXS) to the structural characterization of transmembrane proteins (MPs) in detergent solutions has become a routine procedure at synchrotron BioSAXS beamlines around the world. SAXS provides overall parameters and low resolution shapes of solubilized MPs, but is also meaningfully employed in hybrid modeling procedures that combine scattering data with information provided by high-resolution techniques (eg. macromolecular crystallography, nuclear magnetic resonance and cryo-electron microscopy). Structural modeling of MPs from SAXS data is non-trivial, and the necessary computational procedures require further formalization and facilitation. We propose an automated pipeline integrated with the laboratory-information management system ISPyB, aimed at preliminary SAXS analysis and the first-step reconstruction of MPs in detergent solutions, in order to streamline high-throughput studies, especially at synchrotron beamlines. The pipeline queries an ISPyB database for available a priori information via dedicated services, estimates model-free SAXS parameters and generates preliminary models utilizing either ab initio, high-resolution-based, or mixed/hybrid methods. The results of the automated analysis can be inspected online using the standard ISPyB interface and the estimated modeling parameters may be utilized for further in-depth modeling beyond the pipeline. Examples of the pipeline results for the modelling of the tetrameric alpha-helical membrane channel Aquaporin0 and mechanosensitive channel T2, solubilized by n-Dodecyl β-D-maltoside are presented. We demonstrate how increasing the amount of a priori information improves model resolution and enables deeper insights into the molecular structure of protein-detergent complexes., Published by Macmillan Publishers Limited, part of Springer Nature, [London]

Small-angle X-ray scattering (SAXS) and Nuclear Magnetic Resonance (NMR) are established methods to analyze the structure and structural transitions of biological macromolecules in solution. Both methods are directly applicable to near-native macromolecular solutions and allow one to study structural responses to physical and chemical changes or ligand additions. Whereas SAXS is applied to elucidate overall structure, interactions and flexibility over a wide range of particle sizes, NMR yields atomic resolution detail for moderately sized macromolecules. NMR is arguably the most powerful technique for the experimental analysis of dynamics. The joint application of these two highly complementary techniques provides an extremely useful approach that facilitates comprehensive characterization of biomacromolecular solutions., Highlights • SAXS and NMR are effective and highly complementary techniques in structural biology. • Constraints from SAXS can be readily incorporated in NMR structure calculations. • High resolution NMR models of domains can serve as building blocks for SAXS-based rigid body modeling. • Flexible systems can be well described using ensemble approaches combining SAXS and NMR. • Dynamics studies can be enhanced by combining SAXS and NMR.

The journal of biological chemistry 294(18), 7403 - 7418 (2019). doi:10.1074/jbc.RA119.007847, The urokinase receptor (uPAR) is a founding member of a small protein family with multiple Ly6/uPAR (LU) domains. The motif defining these LU domains contains five plesiotypic disulfide bonds stabilizing its prototypical three-fingered fold having three protruding loops. Notwithstanding the detailed knowledge on structure-function relationships in uPAR, one puzzling enigma remains unexplored. Why does the first LU domain in uPAR (DI) lack one of its consensus disulfide bonds, when the absence of this particular disulfide bond impairs the correct folding of other single LU domain-containing proteins? Here, using a variety of contemporary biophysical methods, we found that reintroducing the two missing half-cystines in uPAR DI caused the spontaneous formation of the corresponding consensus 7–8 LU domain disulfide bond. Importantly, constraints due to this cross-link impaired (i) the binding of uPAR to its primary ligand urokinase and (ii) the flexible interdomain assembly of the three LU domains in uPAR. We conclude that the evolutionary deletion of this particular disulfide bond in uPAR DI may have enabled the assembly of a high-affinity urokinase-binding cavity involving all three LU domains in uPAR. Of note, an analogous neofunctionalization occurred in snake venom α-neurotoxins upon loss of another pair of the plesiotypic LU domain half-cystines. In summary, elimination of the 7–8 consensus disulfide bond in the first LU domain of uPAR did have significant functional and structural consequences., Published by Soc., Bethesda, Md.

Summary The regulation of many protein kinases by binding to calcium/calmodulin connects two principal mechanisms in signaling processes: protein phosphorylation and responses to dose- and time-dependent calcium signals. We used the calcium/calmodulin-dependent members of the death-associated protein kinase (DAPK) family to investigate the role of a basic DAPK signature loop near the kinase active site. In DAPK2, this loop comprises a novel dimerization-regulated calcium/calmodulin-binding site, in addition to a well-established calcium/calmodulin site in the C-terminal autoregulatory domain. Unexpectedly, impairment of the basic loop interaction site completely abolishes calcium/calmodulin binding and DAPK2 activity is reduced to a residual level, indicative of coupled binding to the two sites. This contrasts with the generally accepted view that kinase calcium/calmodulin interactions are autonomous of the kinase catalytic domain. Our data establish an intricate model of multi-step kinase activation and expand our understanding of how calcium binding connects with other mechanisms involved in kinase activity regulation., Graphical Abstract, Highlights • Members of the DAPK family share a specific basic-loop-mediated dimerization motif • DAPK2 contains a kinase-mediated and dimerization-regulated CaM-binding site • Autoregulatory segment CaM binding depends on kinase-mediated CaM binding • DAPK2 activity is regulated by coupled CaM binding to two different sites, Simon et al. show that human death-associated protein kinase activity is regulated by coupled binding of calcium/calmodulin to two distinct sites, a novel kinase-mediated site and an autoregulatory segment-mediated site, and thus establish a new link to the role of calcium in kinase signaling.

Resistance to the last-resort antibiotic colistin is now widespread and new therapeutics are urgently required. We report the first in toto chemical synthesis and pre-clinical evaluation of octapeptins, a class of lipopeptides structurally related to colistin. The octapeptin biosynthetic cluster consisted of three non-ribosomal peptide synthetases (OctA, OctB, and OctC) that produced an amphiphilic antibiotic, octapeptin C4, which was shown to bind to and depolarize membranes. While active against multi-drug resistant (MDR) strains in vitro, octapeptin C4 displayed poor in vivo efficacy, most likely due to high plasma protein binding. Nuclear magnetic resonance solution structures, empirical structure-activity and structure-toxicity models were used to design synthetic octapeptins active against MDR and extensively drug-resistant (XDR) bacteria. The scaffold was then subtly altered to reduce plasma protein binding, while maintaining activity against MDR and XDR bacteria. In vivo efficacy was demonstrated in a murine bacteremia model with a colistin-resistant P. aeruginosa clinical isolate.

Summary Saposin-derived lipid nanoparticles (SapNPs) are a new alternative tool for membrane protein reconstitution. Here we demonstrate the potential and advantages of SapNPs. We show that SapA has the lowest lipid specificity for SapNP formation. These nanoparticles are modular and offer a tunable range of size and composition depending on the stoichiometric ratio of lipid and saposin components. They are stable and exhibit features typical of lipid-bilayer systems. Our data suggest that SapNPs are versatile and can adapt to membrane proteins of various sizes and architectures. Using SapA and various types of lipids we could reconstitute membrane proteins of different transmembrane cross-sectional areas (from 14 to 56 transmembrane α helices). SapNP-reconstituted proteins bound their respective ligands and were more heat stable compared with the detergent-solubilized form. Moreover, SapNPs encircle membrane proteins in a compact way, allowing structural investigations of small membrane proteins in a detergent-free environment using small-angle X-ray scattering., Graphical Abstract, Highlights • SapA shows the lowest lipid specificity for SapNP formation • SapNPs are versatile and can adapt to MPs of various sizes and architectures • SapNP-reconstituted MPs are more stable than in detergent • SapNPs encapsulate MPs in a compact manner, Saposin-derived lipid nanoparticles (SapNPs) are an alternative tool for membrane protein (MP) reconstitution. Flayhan et al. characterize these nanoparticles, showing that they are versatile and can adapt to MPs of various sizes and architectures. SapNP-reconstituted MPs can be treated as soluble proteins and used for structural and functional studies.

Structural studies of integral membrane proteins (IMPs) are challenging, as many of them are inactive or insoluble in the absence of a lipid environment. Here, we describe an approach making use of fractionally deuterium labeled "stealth carrier" nanodiscs that are effectively invisible to low-resolution neutron diffraction and enable structural studies of IMPs in a lipidic native-like solution environment. We illustrate the potential of the method in a joint small-angle neutron scattering (SANS) and X-ray scattering (SAXS) study of the ATP-binding cassette (ABC) transporter protein MsbA solubilized in the stealth nanodiscs. The data allow for a direct observation of the signal from the solubilized protein without contribution from the surrounding lipid nanodisc. Not only the overall shape but also differences between conformational states of MsbA can be reliably detected from the scattering data, demonstrating the sensitivity of the approach and its general applicability to structural studies of IMPs.

The urokinase-type plasminogen activator receptor (uPAR) provides a rendezvous between proteolytic degradation of the extracellular matrix and integrin-mediated adhesion to vitronectin. These processes are, however, tightly linked because the high affinity binding of urokinase regulates the binding of uPAR to matrix-embedded vitronectin. Although crystal structures exist to define the corresponding static bi- and trimolecular receptor complexes, it is evident that the dynamic property of uPAR plays a decisive role in its function. In the present study, we combine small angle x-ray scattering, hydrogen-deuterium exchange, and surface plasmon resonance to develop a structural model describing the allosteric regulation of uPAR. We show that the flexibility of its N-terminal domain provides the key for understanding this allosteric mechanism. Importantly, our model has direct implications for understanding uPAR-assisted cell adhesion and migration as well as for translational research, including targeted intervention therapy and non-invasive tumor imaging in vivo.

The journal of biological chemistry 291(28), 14430 - 14446(2016). doi:10.1074/jbc.M116.730382, Glycyl tRNA synthetase (GlyRS) provides a unique case among class II aminoacyl tRNA synthetases (aaRSs), with two clearly widespread types of enzymes: a dimeric ($\alpha_2$) species present in some bacteria, archaea, and eukaryotes, and a heterotetrameric form ($\alpha_{2}\beta_2$) present in most bacteria. Although the differences between both types of GlyRS at the anticodon binding domain level are evident, the extent and implications of the variations in the catalytic domain have not been described and it is unclear whether the mechanism of amino acid recognition is also dissimilar. Here we show that the a-subunit of the $\alpha_{2}\beta_2$ GlyRS from the bacterium Aquifex aeolicus is able to perform the first step of the aminoacylation reaction, which involves the activation of the amino acid with ATP. The crystal structure of the a-subunit in the complex with an analog of glycyl adenylate at 2.8 A resolution presents a conformational arrangement that properly positions the cognate amino acid. This work shows that glycine is recognized by a subset of different residues in the two types of GlyRS. A structural and sequence analysis of class II catalytic domains shows that bacterial GlyRS is closely related to AlaRS, which led us to define a new subclassification of these ancient enzymes and to propose an evolutionary path of $\alpha_{2}\beta_2$ GlyRS, convergent with $\alpha_{2}$ GlyRS and divergent from AlaRS, thus providing a possible explanation for the puzzling existence of two proteins sharing the same fold and function, but not a common ancestor., Published by Soc., Bethesda, Md.

Bacteriophages produce endolysins, which lyse the bacterial host cell to release newly produced virions. The timing of lysis is regulated and is thought to involve the activation of a molecular switch. We present a crystal structure of the activated endolysin CTP1L that targets Clostridium tyrobutyricum, consisting of a complex between the full-length protein and an N-terminally truncated C-terminal cell wall binding domain (CBD). The truncated CBD is produced through an internal translation start site within the endolysin gene. Mutants affecting the internal translation site change the oligomeric state of the endolysin and reduce lytic activity. The activity can be modulated by reconstitution of the full-length endolysin-CBD complex with free CBD. The same oligomerization mechanism applies to the CD27L endolysin that targets Clostridium difficileand the CS74L endolysin that targets Clostridium sporogenes. When the CTP1L endolysin gene is introduced into the commensal bacterium Lactococcus lactis, the truncated CBD is also produced, showing that the alternative start codon can be used in other bacterial species. The identification of a translational switch affecting oligomerization presented here has implications for the design of effective endolysins for the treatment of bacterial infections. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The NuRD (nucleosome remodeling and deacetylase) complex serves as a crucial epigenetic regulator of cell differentiation, proliferation, and hematopoietic development by coupling the deacetylation and demethylation of histones, nucleosome mobilization, and the recruitment of transcription factors. The core nucleosome remodeling function of the mammalian NuRD complex is executed by the helicase-domain-containing ATPase CHD4 (Mi-2β) subunit, which also contains N-terminal plant homeodomain (PHD) and chromo domains. The mode of regulation of chromatin remodeling by CHD4 is not well understood, nor is the role of its PHD and chromo domains. Here, we use small-angle X-ray scattering, nucleosome binding ATPase and remodeling assays, limited proteolysis, cross-linking, and tandem mass spectrometry to propose a three-dimensional structural model describing the overall shape and domain interactions of CHD4 and discuss the relevance of these for regulating the remodeling of chromatin by the NuRD complex., Graphical Abstract Highlights ► The ATPase CHD4 mediates nucleosome remodeling by the NuRD complex. ► We present a three-dimensional small-angle X-ray scattering model of CHD4 and define its interdomain interactions. ► Cross-linking and limited proteolysis studies validate our model. ► Functional and binding assays suggest a regulatory role for the PHD and chromo domains.

The first eight and the last two of 20 complement control protein (CCP) modules within complement factor H (fH) encompass binding sites for C3b and polyanionic carbohydrates. These binding sites cooperate self-surface selectively to prevent C3b amplification, thus minimising complement-mediated damage to host. Intervening fH CCPs, apparently devoid of such recognition sites, are proposed to play a structural role. One suggestion is that the generally small CCPs 10–15, connected by longer-than-average linkers, act as a flexible tether between the two functional ends of fH; another is that the long linkers induce a 180° bend in the middle of fH. To test these hypotheses, we determined the NMR-derived structure of fH12–13 consisting of module 12, shown here to have an archetypal CCP structure, and module 13, which is uniquely short and features a laterally protruding helix-like insertion that contributes to a prominent electropositive patch. The unusually long fH12–13 linker is not flexible. It packs between the two CCPs that are not folded back on each other but form a shallow vee shape; analytical ultracentrifugation and X-ray scattering supported this finding. These two techniques additionally indicate that flanking modules (within fH11–14 and fH10–15) are at least as rigid and tilted relative to neighbours as are CCPs 12 and 13 with respect to one another. Tilts between successive modules are not unidirectional; their principal axes trace a zigzag path. In one of two arrangements for CCPs 10–15 that fit well with scattering data, CCP 14 is folded back onto CCP 13. In conclusion, fH10–15 forms neither a flexible tether nor a smooth bend. Rather, it is compact and has embedded within it a CCP module (CCP 13) that appears to be highly specialised given both its deviant structure and its striking surface charge distribution. A passive, purely structural role for this central portion of fH is unlikely.

FYVE domain proteins play key roles in regulating membrane traffic in eukaryotic cells. The FYVE domain displays a remarkable specificity for the head group of the target lipid, phosphatidylinositol 3-phosphate (PtdIns[3]P). We have identified five putative FYVE domain proteins in the genome of the protozoan parasite Leishmania major, three of which are predicted to contain a functional PtdIns(3)P-binding site. The FYVE domain of one of these proteins, LmFYVE-1, bound PtdIns(3)P in liposome-binding assays and targeted GFP to acidified late endosomes/lysosomes in mammalian cells. The high-resolution solution structure of its N-terminal FYVE domain (LmFYVE-1[1–79]) was solved by nuclear magnetic resonance. Functionally significant clusters of residues of the LmFYVE-1 domain involved in PtdIns(3)P binding and dependence on low pH for tight binding were identified. This structure is the first trypanosomatid membrane trafficking protein to be determined and has been refined to high precision and accuracy using residual dipolar couplings.

The AMP-activated protein kinase (AMPK) contains a carbohydrate-binding module (beta1-CBM) that is conserved from yeast to mammals. Beta1-CBM has been shown to localize AMPK to glycogen in intact cells and in vitro. Here we use Nuclear Magnetic Resonance spectroscopy to investigate oligosaccharide binding to 15N labelled beta1-CBM. We find that beta1-CBM shows greatest affinity to carbohydrates of greater than five glucose units joined via alpha,1-->4 glycosidic linkages with a single, but not multiple, glucose units in an alpha,1-->6 branch. The near identical chemical shift profile for all oligosaccharides whether cyclic or linear suggest a similar binding conformation and confirms the presence of a single carbohydrate-binding site.

Plasma apolipoproteins show alpha-helical structure in the lipid-bound state and limited conformational stability in the absence of lipid. This structural instability of lipid-free apolipoproteins may account for the high propensity of apolipoproteins to aggregate and accumulate in disease-related amyloid deposits. Here, we explore the properties of amyloid fibrils formed by apolipoproteins using human apolipoprotein (apo) C-II as a model system. Hydrogen-deuterium exchange and NMR spectroscopy of apoC-II fibrils revealed core regions between residues 19-37 and 57-74 with reduced amide proton exchange rates compared to monomeric apoC-II. The C-terminal core region was also identified by partial proteolysis of apoC-II amyloid fibrils using endoproteinase GluC and proteinase K. Complete tryptic hydrolysis of apoC-II fibrils followed by centrifugation yielded a single peptide in the pellet fraction identified using mass spectrometry as apoC-II(56-76). Synthetic apoC-II(56-76) readily formed fibrils, albeit with a different morphology and thioflavinT fluorescence yield compared to full-length apoC-II. Studies with smaller peptides narrowed this fibril-forming core to a region within residues 60-70. We postulate that the ability of apoC-II(60-70) to independently form amyloid fibrils drives fibril formation by apoC-II. These specific amyloid-forming regions within apolipoproteins may underlie the propensity of apolipoproteins and their peptide derivatives to accumulate in amyloid deposits in vivo.

The journal of biological chemistry 290(5), 2644 - 2658(2015). doi:10.1074/jbc.M114.587667, Ethylene initiates important aspects of plant growth and development through disulfide-linked receptor dimers located in the endoplasmic reticulum. The receptors feature a small transmembrane, ethylene binding domain followed by a large cytosolic domain, which serves as a scaffold for the assembly of large molecular weight complexes of different ethylene receptors and other cellular participants of the ethylene signaling pathway. Here we report the crystallographic structures of the ethylene receptor 1 (ETR1) catalytic ATP-binding and the ethylene response sensor 1 dimerization histidine phosphotransfer (DHp) domains and the solution structure of the entire cytosolic domain of ETR1, all from Arabidopsis thaliana. The isolated dimeric ethylene response sensor 1 DHp domain is asymmetric, the result of different helical bending angles close to the conserved His residue. The structures of the catalytic ATP-binding, DHp, and receiver domains of ethylene receptors and of a homologous, but dissimilar, GAF domain were refined against experimental small angle x-ray scattering data, leading to a structural model of the entire cytosolic domain of the ethylene receptor 1. The model illustrates that the cytosolic domain is shaped like a dumbbell and that the receiver domain is flexible and assumes a position different from those observed in prokaryotic histidine kinases. Furthermore the cytosolic domain of ETR1 plays a key role, interacting with all other receptors and several participants of the ethylene signaling pathway. Our model, therefore, provides the first step toward a detailed understanding of the molecular mechanics of this important signal transduction process in plants., Published by Soc., Bethesda, Md.

To survive and replicate within the human host, malaria parasites must invade erythrocytes. Invasion can be mediated by the P. falciparum reticulocyte-binding homologue protein 4 (PfRh4) on the merozoite surface interacting with complement receptor type 1 (CR1, CD35) on the erythrocyte membrane. The PfRh4 attachment site lies within the three N-terminal complement control protein modules (CCPs 1-3) of CR1, which intriguingly also accommodate binding and regulatory sites for the key complement activation-specific proteolytic products, C3b and C4b. One of these regulatory activities is decay-accelerating activity. Although PfRh4 does not impact C3b/C4b binding, it does inhibit this convertase disassociating capability. Here, we have employed ELISA, co-immunoprecipitation, and surface plasmon resonance to demonstrate that CCP 1 contains all the critical residues for PfRh4 interaction. We fine mapped by homologous substitution mutagenesis the PfRh4-binding site on CCP 1 and visualized it with a solution structure of CCPs 1-3 derived by NMR and small angle x-ray scattering. We cross-validated these results by creating an artificial PfRh4-binding site through substitution of putative PfRh4-interacting residues from CCP 1 into their homologous positions within CCP 8; strikingly, this engineered binding site had an approximate to 30-fold higher affinity for PfRh4 than the native one in CCP 1. These experiments define a candidate site on CR1 by which P. falciparum merozoites gain access to human erythrocytes in a non-sialic acid-dependent pathway of merozoite invasion.

Ribulose-bisphosphate carboxylase/oxygenase (Rubisco) activase uses the energy from ATP hydrolysis to remove tight binding inhibitors from Rubisco, thus playing a key role in regulating photosynthesis in plants. Although several structures have recently added much needed structural information for different Rubisco activase enzymes, the arrangement of these subunits in solution remains unclear. In this study, we use a variety of techniques to show that Rubisco activase forms a wide range of structures in solution, ranging from monomers to much higher order species, and that the distribution of these species is highly dependent on protein concentration. The data support a model in which Rubisco activase forms an open spiraling structure rather than a closed hexameric structure. At protein concentrations of 1 μm, corresponding to the maximal activity of the enzyme, Rubisco activase has an oligomeric state of 2–4 subunits. We propose a model in which Rubisco activase requires at least 1 neighboring subunit for hydrolysis of ATP.

The 155-kDa plasma glycoprotein factor H (FH), which consists of 20 complement control protein (CCP) modules, protects self-tissue but not foreign organisms from damage by the complement cascade. Protection is achieved by selective engagement of FH, via CCPs 1–4, CCPs 6–8 and CCPs 19–20, with polyanion-rich host surfaces that bear covalently attached, activation-specific, fragments of complement component C3. The role of intervening CCPs 9–18 in this process is obscured by lack of structural knowledge. We have concatenated new high-resolution solution structures of overlapping recombinant CCP pairs, 10–11 and 11–12, to form a three-dimensional structure of CCPs 10–12 and validated it by small-angle X-ray scattering of the recombinant triple‐module fragment. Superimposing CCP 12 of this 10–12 structure with CCP 12 from the previously solved CCP 12–13 structure yielded an S-shaped structure for CCPs 10–13 in which modules are tilted by 80–110° with respect to immediate neighbors, but the bend between CCPs 10 and 11 is counter to the arc traced by CCPs 11–13. Including this four-CCP structure in interpretation of scattering data for the longer recombinant segments, CCPs 10–15 and 8–15, implied flexible attachment of CCPs 8 and 9 to CCP 10 but compact and intimate arrangements of CCP 14 with CCPs 12, 13 and 15. Taken together with difficulties in recombinant production of module pairs 13–14 and 14–15, the aberrant structure of CCP 13 and the variability of 13–14 linker sequences among orthologues, a structural dependency of CCP 14 on its neighbors is suggested; this has implications for the FH mechanism., Graphical abstract Highlights ► The 20-CCP‐module human protein FH prevents complement-mediated tissue damage. ► NMR structures of CCPs 10–11 and 11–12 suggest that this region enhances flexional strength of FH. ► Concatenating bi-modules helps interpret small‐angle X‐ray scattering data, revealing highly compacted arrangement of CCPs 13, 14 and 15. ► Apparent structural dependency of CCP 14 on neighbors could provide a switch between ordered and flexible FH architectures.

Complement receptor-type 1 (CR1, CD35) is the immune-adherence receptor, a complement regulator, and an erythroid receptor for Plasmodium falciparum during merozoite invasion and subsequent rosette formation involving parasitized and non-infected erythrocytes. The non-uniform geographical distribution of Knops blood group CR1 alleles Sl1/2 and McC(a/b) may result from selective pressures exerted by differential exposure to infectious hazards. Here, four variant short recombinant versions of CR1 were produced and analyzed, focusing on complement control protein modules (CCPs) 15-25 of its ectodomain. These eleven modules encompass a region (CCPs 15-17) key to rosetting, opsonin recognition and complement regulation, as well as the Knops blood group polymorphisms in CCPs 24-25. All four CR1 15-25 variants were monomeric and had similar axial ratios. Modules 21 and 22, despite their double-length inter-modular linker, did not lie side-by-side so as to stabilize a bent-back architecture that would facilitate cooperation between key functional modules and Knops blood group antigens. Indeed, the four CR1 15-25 variants had virtually indistinguishable affinities for immobilized complement fragments C3b (K(D) = 0.8-1.1 µM) and C4b (K(D) = 5.0-5.3 µM). They were all equally good co-factors for factor I-catalysed cleavage of C3b and C4b, and they bound equally within a narrow affinity range, to immobilized C1q. No differences between the variants were observed in assays for inhibition of erythrocyte invasion by P. falciparum or for rosette disruption. Neither differences in complement-regulatory functionality, nor interactions with P. falciparum proteins tested here, appear to have driven the non-uniform geographic distribution of these alleles.

Complement factor H (FH) attenuates C3b molecules tethered via their thioester domains to selfsurfaces and thereby protects host tissues. FH is a cofactor for initial C3b proteolysis that ultimately yields a surface-attached fragment (C3d), corresponding to the thioester domain. We used NMR and X-ray crystallography to study the C3d:FH19–20 complex in atomic detail. NMR further identified glycosaminoglycan-binding residues in FH module 20 of the C3d:FH19–20 complex. Mutagenesis justified the merging of the C3d:FH19–20 structure with an existing C3b:FH1–4 crystal structure. The merged structure was concatenated with the available FH6–8 crystal structure and new SAXS-derived FH1–4, FH8–15 and FH15–19 envelopes. The combined data suggests a bent-back FH molecule, binding via its termini to two sites on one C3b molecule and simultaneously to adjacent polyanionic host-surface markers. The ~30 proteins of the complement system cooperate in a vital contribution to innate immunity 1 . The complement system is important for clearance of immune complexes and cellular debris, and for augmenting cell-based immunity, but is most famous for rapid targeting and elimination of pathogens. Host tissues may also sustain complement-mediated damage; several diseases, including age-related macular degeneration, atypical hemolytic

The Tom20 and Tom22 receptor subunits of the TOM (translocase of the outer mitochondrial membrane) complex recognize N-terminal presequences of proteins that are to be imported into the mitochondrion. In plants, Tom20 is C-terminally anchored in the mitochondrial membrane, whereas Tom20 is N-terminally anchored in animals and fungi. Furthermore, the cytosolic domain of Tom22 in plants is smaller than its animal/fungal counterpart and contains fewer acidic residues. Here, NMR spectroscopy was used to explore presequence interactions with the cytosolic regions of receptors from the plant Arabidopsis thaliana and the fungus Saccharomyces cerevisiae (i.e., AtTom20, AtTom22, and ScTom22). It was found that AtTom20 possesses a discontinuous bidentate hydrophobic binding site for presequences. The presequences on plant mitochondrial proteins comprise two or more hydrophobic binding regions to match this bidentate site. NMR data suggested that while these presequences bind to ScTom22, they do not bind to AtTom22. AtTom22, however, binds to AtTom20 at the same binding site as presequences, suggesting that this domain competes with the presequences of imported proteins, thereby enabling their progression along the import pathway.

The refinement of protein structures determined by nuclear magnetic resonance spectroscopy against database potentials of mean force allows for the exclusion of unfavourable conformations of the protein backbone during a structure calculation, resulting in protein structures with a marked improvement in Ramachandran statistics. In this communication, we use multiple sets of residual dipolar couplings as quality assessment criteria for several proteins and show that not only do the Ramachandran and structural quality statistics improve, but a significant improvement in the accuracy of structures is achieved upon refinement.