DnaK response to expression of protein mutants is dependent on translation rate and stability.

Chaperones play a central part in the quality control system in cells by clearing misfolded and aggregated proteins. The chaperone DnaK acts as a sensor for molecular stress by recognising short hydrophobic stretches of misfolded proteins. As the level of unfolded protein is a function of protein stability, we hypothesised that the level of DnaK response upon overexpression of recombinant proteins would be correlated to stability. Using a set of mutants of the λ-repressor with varying thermal stabilities and a fluorescent reporter system, the effect of stability on DnaK response and protein abundance was investigated. Our results demonstrate that the initial DnaK response is largely dependent on protein synthesis rate but as the recombinantly expressed protein accumulates and homeostasis is approached the response correlates strongly with stability. Furthermore, we observe a large degree of cell-cell variation in protein abundance and DnaK response in more stable proteins.

Potent antibody lineage against malaria transmission elicited by human vaccination with Pfs25.

Transmission-blocking vaccines have the potential to be key contributors to malaria elimination. Such vaccines elicit antibodies that inhibit parasites during their development in Anopheles mosquitoes, thus breaking the cycle of transmission. To date, characterization of humoral responses to Plasmodium falciparum transmission-blocking vaccine candidate Pfs25 has largely been conducted in pre-clinical models. Here, we present molecular analyses of human antibody responses generated in a clinical trial evaluating Pfs25 vaccination. From a collection of monoclonal antibodies with transmission-blocking activity, we identify the most potent transmission-blocking antibody yet described against Pfs25; 2544. The interactions of 2544 and three other antibodies with Pfs25 are analyzed by crystallography to understand structural requirements for elicitation of human transmission-blocking responses. Our analyses provide insights into Pfs25 immunogenicity and epitope potency, and detail an affinity maturation pathway for a potent transmission-blocking antibody in humans. Our findings can be employed to guide the design of improved malaria transmission-blocking vaccines.

Automatically Fixing Errors in Glycoprotein Structures with Rosetta.

Recent advances in single-particle cryo-electron microscopy (cryoEM) have resulted in determination of an increasing number of protein structures with resolved glycans. However, existing protocols for the refinement of glycoproteins at low resolution have failed to keep up with these advances. As a result, numerous deposited structures contain glycan stereochemical errors. Here, we describe a Rosetta-based approach for both cryoEM and X-ray crystallography refinement of glycoproteins that is capable of correcting conformational and configurational errors in carbohydrates. Building upon a previous Rosetta framework, we introduced additional features and score terms enabling automatic detection, setup, and refinement of glycan-containing structures. We benchmarked this approach using 12 crystal structures and showed that glycan geometries can be automatically improved while maintaining good fit to the crystallographic data. Finally, we used this method to refine carbohydrates of the human coronavirus NL63 spike glycoprotein and of an HIV envelope glycoprotein, demonstrating its usefulness for cryoEM refinement.

Glycoengineering HIV-1 Env creates 'supercharged' and 'hybrid' glycans to increase neutralizing antibody potency, breadth and saturation.

The extensive glycosylation of HIV-1 envelope (Env) glycoprotein leaves few glycan-free holes large enough to admit broadly neutralizing antibodies (bnAb). Consequently, most bnAbs must inevitably make some glycan contacts and avoid clashes with others. To investigate how Env glycan maturation regulates HIV sensitivity to bnAbs, we modified HIV-1 pseudovirus (PV) using various glycoengineering (GE) tools. Promoting the maturation of α-2,6 sialic acid (SA) glycan termini increased PV sensitivity to two bnAbs that target the V2 apex and one to the interface between Env surface gp120 and transmembrane gp41 subunits, typically by up to 30-fold. These effects were reversible by incubating PV with neuraminidase. The same bnAbs were unusually potent against PBMC-produced HIV-1, suggesting similar α-2,6 hypersialylated glycan termini may occur naturally. Overexpressing ß-galactosyltransferase during PV production replaced complex glycans with hybrid glycans, effectively 'thinning' trimer glycan coverage. This increased PV sensitivity to some bnAbs but ablated sensitivity to one bnAb that depends on complex glycans. Other bnAbs preferred small glycans or galactose termini. For some bnAbs, the effects of GE were strain-specific, suggesting that GE had context-dependent effects on glycan clashes. GE was also able to increase the percent maximum neutralization (i.e. saturation) by some bnAbs. Indeed, some bnAb-resistant strains became highly sensitive with GE-thus uncovering previously unknown bnAb breadth. As might be expected, the activities of bnAbs that recognize glycan-deficient or invariant oligomannose epitopes were largely unaffected by GE. Non-neutralizing antibodies were also unaffected by GE, suggesting that trimers remain compact. Unlike mature bnAbs, germline-reverted bnAbs avoided or were indifferent to glycans, suggesting that glycan contacts are acquired as bnAbs mature. Together, our results suggest that glycovariation can greatly impact neutralization and that knowledge of the optimal Env glycoforms recognized by bnAbs may assist rational vaccine design.

Molecular definition of multiple sites of antibody inhibition of malaria transmission-blocking vaccine antigen Pfs25.

The Plasmodium falciparum Pfs25 protein (Pfs25) is a leading malaria transmission-blocking vaccine antigen. Pfs25 vaccination is intended to elicit antibodies that inhibit parasite development when ingested by Anopheles mosquitoes during blood meals. The Pfs25 three-dimensional structure has remained elusive, hampering a molecular understanding of its function and limiting immunogen design. We report six crystal structures of Pfs25 in complex with antibodies elicited by immunization via Pfs25 virus-like particles in human immunoglobulin loci transgenic mice. Our structural findings reveal the fine specificities associated with two distinct immunogenic sites on Pfs25. Importantly, one of these sites broadly overlaps with the epitope of the well-known 4B7 mouse antibody, which can be targeted simultaneously by antibodies that target a non-overlapping site to additively increase parasite inhibition. Our molecular characterization of inhibitory antibodies informs on the natural disposition of Pfs25 on the surface of ookinetes and provides the structural blueprints to design next-generation immunogens.

Three in One: Temperature, Solvent and Catalytic Stability by Engineering the Cofactor-Binding Element of Amine Transaminase.

Amine transaminase (ATA) catalyse enantioselectively the direct amination of ketones, but insufficient stability during catalysis limits their industrial applicability. Recently, we revealed that ATAs suffer from substrate-induced inactivation mechanism involving dissociation of the enzyme-cofactor intermediate. Here, we report on engineering the cofactor-ring-binding element, which also shapes the active-site entrance. Only two point mutations in this motif improved temperature and catalytic stability in both biphasic media and organic solvent. Thermodynamic analysis revealed a higher melting point for the enzyme-cofactor intermediate. The high cofactor affinity eliminates the need for pyridoxal 5'-phosphate supply, thus making large-scale reactions more cost effective. This is the first report on stabilising a tetrameric ATA by mutating a single structural element. As this structural "hotspot" is a common feature of other transaminases it could serve as a general engineering target.

Crystal structure of human chondroadherin: solving a difficult molecular-replacement problem using de novo models.

Chondroadherin (CHAD) is a cartilage matrix protein that mediates the adhesion of isolated chondrocytes. Its protein core is composed of 11 leucine-rich repeats (LRR) flanked by cysteine-rich domains. CHAD makes important interactions with collagen as well as with cell-surface heparin sulfate proteoglycans and α2ß1 integrins. The integrin-binding site is located in a region of hitherto unknown structure at the C-terminal end of CHAD. Peptides based on the C-terminal human CHAD (hCHAD) sequence have shown therapeutic potential for treating osteoporosis. This article describes a still-unconventional structure solution by phasing with de novo models, the first of a ß-rich protein. Structure determination of hCHAD using traditional, though nonsystematic, molecular replacement was unsuccessful in the hands of the authors, possibly owing to a combination of low sequence identity to other LRR proteins, four copies in the asymmetric unit and weak translational pseudosymmetry. However, it was possible to solve the structure by generating a large number of de novo models for the central LRR domain using Rosetta and multiple parallel molecular-replacement attempts using AMPLE. The hCHAD structure reveals an ordered C-terminal domain belonging to the LRRCT fold, with the integrin-binding motif (WLEAK) being part of a regular α-helix, and suggests ways in which experimental therapeutic peptides can be improved. The crystal structure itself and docking simulations further support that hCHAD dimers form in a similar manner to other matrix LRR proteins.

Automated de novo phasing and model building of coiled-coil proteins.

Models generated by de novo structure prediction can be very useful starting points for molecular replacement for systems where suitable structural homologues cannot be readily identified. Protein-protein complexes and de novo-designed proteins are examples of systems that can be challenging to phase. In this study, the potential of de novo models of protein complexes for use as starting points for molecular replacement is investigated. The approach is demonstrated using homomeric coiled-coil proteins, which are excellent model systems for oligomeric systems. Despite the stereotypical fold of coiled coils, initial phase estimation can be difficult and many structures have to be solved with experimental phasing. A method was developed for automatic structure determination of homomeric coiled coils from X-ray diffraction data. In a benchmark set of 24 coiled coils, ranging from dimers to pentamers with resolutions down to 2.5 Å, 22 systems were automatically solved, 11 of which had previously been solved by experimental phasing. The generated models contained 71-103% of the residues present in the deposited structures, had the correct sequence and had free R values that deviated on average by 0.01 from those of the respective reference structures. The electron-density maps were of sufficient quality that only minor manual editing was necessary to produce final structures. The method, named CCsolve, combines methods for de novo structure prediction, initial phase estimation and automated model building into one pipeline. CCsolve is robust against errors in the initial models and can readily be modified to make use of alternative crystallographic software. The results demonstrate the feasibility of de novo phasing of protein-protein complexes, an approach that could also be employed for other small systems beyond coiled coils.

Exploring alternate states and oligomerization preferences of coiled-coils by de novo structure modeling.

Homomeric coiled-coils can self-assemble into a wide range of structural states with different helix topologies and oligomeric states. In this study, we have combined de novo structure modeling with stability calculations to simultaneously predict structure and oligomeric states of homomeric coiled-coils. For dimers an asymmetric modeling protocol was developed. Modeling without symmetry constraints showed that backbone asymmetry is important for the formation of parallel dimeric coiled-coils. Collectively, our results demonstrate that high-resolution structure of coiled-coils, as well as parallel and antiparallel orientations of dimers and tetramers, can be accurately predicted from sequence. De novo modeling was also used to generate models of competing oligomeric states, which were used to compare stabilities and thus predict the native stoichiometry from sequence. In a benchmark set of 33 coiled-coil sequences, forming dimers to pentamers, up to 70% of the oligomeric states could be correctly predicted. The calculations demonstrated that the free energy of helix folding could be an important factor for determining stability and oligomeric state of homomeric coiled-coils. The computational methods developed here should be broadly applicable to studies of sequence-structure relationships in coiled-coils and the design of higher order assemblies with improved oligomerization specificity.

Computational design of a leucine-rich repeat protein with a predefined geometry.

Structure-based protein design offers a possibility of optimizing the overall shape of engineered binding scaffolds to match their targets better. We developed a computational approach for the structure-based design of repeat proteins that allows for adjustment of geometrical features like length, curvature, and helical twist. By combining sequence optimization of existing repeats and de novo design of capping structures, we designed leucine-rich repeats (LRRs) from the ribonuclease inhibitor (RI) family that assemble into structures with a predefined geometry. The repeat proteins were built from self-compatible LRRs that are designed to interact to form highly curved and planar assemblies. We validated the geometrical design approach by engineering a ring structure constructed from 10 self-compatible repeats. Protein design can also be used to increase our structural understanding of repeat proteins. We use our design constructs to demonstrate that buried Cys play a central role for stability and folding cooperativity in RI-type LRR proteins. The computational procedure presented here may be used to develop repeat proteins with various geometrical shapes for applications where greater control of the interface geometry is desired.

Pathogen specific, IRF3-dependent signaling and innate resistance to human kidney infection.

The mucosal immune system identifies and fights invading pathogens, while allowing non-pathogenic organisms to persist. Mechanisms of pathogen/non-pathogen discrimination are poorly understood, as is the contribution of human genetic variation in disease susceptibility. We describe here a new, IRF3-dependent signaling pathway that is critical for distinguishing pathogens from normal flora at the mucosal barrier. Following uropathogenic E. coli infection, Irf3(-/-) mice showed a pathogen-specific increase in acute mortality, bacterial burden, abscess formation and renal damage compared to wild type mice. TLR4 signaling was initiated after ceramide release from glycosphingolipid receptors, through TRAM, CREB, Fos and Jun phosphorylation and p38 MAPK-dependent mechanisms, resulting in nuclear translocation of IRF3 and activation of IRF3/IFNß-dependent antibacterial effector mechanisms. This TLR4/IRF3 pathway of pathogen discrimination was activated by ceramide and by P-fimbriated E. coli, which use ceramide-anchored glycosphingolipid receptors. Relevance of this pathway for human disease was supported by polymorphic IRF3 promoter sequences, differing between children with severe, symptomatic kidney infection and children who were asymptomatic bacterial carriers. IRF3 promoter activity was reduced by the disease-associated genotype, consistent with the pathology in Irf3(-/-) mice. Host susceptibility to common infections like UTI may thus be strongly influenced by single gene modifications affecting the innate immune response.