Plasticity of the binding pocket in peptide transporters underpins promiscuous substrate recognition.

Proton-dependent oligopeptide transporters (POTs) are promiscuous transporters of the major facilitator superfamily that constitute the main route of entry for a wide range of dietary peptides and orally administrated peptidomimetic drugs. Given their clinical and pathophysiological relevance, several POT homologs have been studied extensively at the structural and molecular level. However, the molecular basis of recognition and transport of diverse peptide substrates has remained elusive. We present 14 X-ray structures of the bacterial POT DtpB in complex with chemically diverse di- and tripeptides, providing novel insights into the plasticity of the conserved central binding cavity. We analyzed binding affinities for more than 80 peptides and monitored uptake by a fluorescence-based transport assay. To probe whether all 8400 natural di- and tripeptides can bind to DtpB, we employed state-of-the-art molecular docking and machine learning and conclude that peptides with compact hydrophobic residues are the best DtpB binders.

StarMap: a user-friendly workflow for Rosetta-driven molecular structure refinement.

Cryogenic electron microscopy (cryo-EM) data represent density maps of macromolecular systems at atomic or near-atomic resolution. However, building and refining 3D atomic models by using data from cryo-EM maps is not straightforward and requires significant hands-on experience and manual intervention. We recently developed StarMap, an easy-to-use interface between the popular structural display program ChimeraX and Rosetta, a powerful molecular modeling engine. StarMap offers a general approach for refining structural models of biological macromolecules into cryo-EM density maps by combining Monte Carlo sampling with local density-guided optimization, Rosetta-based all-atom refinement and real-space B-factor calculations in a straightforward workflow. StarMap includes options for structural symmetry, local refinements and independent model validation. The overall quality of the refinement and the structure resolution is then assessed via analytical outputs, such as magnification calibration (pixel size calibration) and Fourier shell correlations. Z-scores reported by StarMap provide an easily interpretable indicator of the goodness of fit for each residue and can be plotted to evaluate structural models and improve local residue refinements, as well as to identify flexible regions and potentially functional sites in large macromolecular complexes. The protocol requires general computer skills, without the need for coding expertise, because most parts of the workflow can be operated by clicking tabs within the ChimeraX graphical user interface. Time requirements for the model refinement depend on the size and quality of the input data; however, this step can typically be completed within 1 d. The analytical parts of the workflow are completed within minutes.

Immune tolerance against infused FVIII in hemophilia A is mediated by PD-L1+ Tregs.

A major complication of hemophilia A therapy is the development of alloantibodies (inhibitors) that neutralize intravenously administered coagulation factor VIII (FVIII). Immune tolerance induction therapy (ITI) by repetitive FVIII injection can eradicate inhibitors, and thereby reduce morbidity and treatment costs. However, ITI success is difficult to predict and the underlying immunological mechanisms are unknown. Here, we demonstrated that immune tolerance against FVIII under nonhemophilic conditions was maintained by programmed death (PD) ligand 1-expressing (PD-L1-expressing) regulatory T cells (Tregs) that ligated PD-1 on FVIII-specific B cells, causing them to undergo apoptosis. FVIII-deficient mice injected with FVIII lacked such Tregs and developed inhibitors. Using an ITI mouse model, we found that repetitive FVIII injection induced FVIII-specific PD-L1+ Tregs and reengaged removal of inhibitor-forming B cells. We also demonstrated the existence of FVIII-specific Tregs in humans and showed that such Tregs upregulated PD-L1 in patients with hemophilia after successful ITI. Simultaneously, FVIII-specific B cells upregulated PD-1 and became killable by Tregs. In summary, we showed that PD-1-mediated B cell tolerance against FVIII operated in healthy individuals and in patients with hemophilia A without inhibitors, and that ITI reengaged this mechanism. These findings may impact monitoring of ITI success and treatment of patients with hemophilia A.

Helical reconstruction of Salmonella and Shigella needle filaments attached to type 3 basal bodies.

Gram-negative pathogens evolved a syringe-like nanomachine, termed type 3 secretion system, to deliver protein effectors into the cytoplasm of host cells. An essential component of this system is a long helical needle filament that protrudes from the bacterial surface and connects the cytoplasms of the bacterium and the eukaryotic cell. Previous structural research was predominantly focused on reconstituted type 3 needle filaments, which lacked the biological context. In this work we introduce a facile procedure to obtain high-resolution cryo-EM structure of needle filaments attached to the basal body of type 3 secretion systems. We validate our approach by solving the structure of Salmonella PrgI filament and demonstrate its utility by obtaining the first high-resolution cryo-EM reconstruction of Shigella MxiH filament. Our work paves the way to systematic structural characterization of attached type 3 needle filaments in the context of mutagenesis studies, protein structural evolution and drug development.

Substrate-engaged type III secretion system structures reveal gating mechanism for unfolded protein translocation.

Many bacterial pathogens rely on virulent type III secretion systems (T3SSs) or injectisomes to translocate effector proteins in order to establish infection. The central component of the injectisome is the needle complex which assembles a continuous conduit crossing the bacterial envelope and the host cell membrane to mediate effector protein translocation. However, the molecular principles underlying type III secretion remain elusive. Here, we report a structure of an active Salmonella enterica serovar Typhimurium needle complex engaged with the effector protein SptP in two functional states, revealing the complete 800Å-long secretion conduit and unraveling the critical role of the export apparatus (EA) subcomplex in type III secretion. Unfolded substrates enter the EA through a hydrophilic constriction formed by SpaQ proteins, which enables side chain-independent substrate transport. Above, a methionine gasket formed by SpaP proteins functions as a gate that dilates to accommodate substrates while preventing leaky pore formation. Following gate penetration, a moveable SpaR loop first folds up to then support substrate transport. Together, these findings establish the molecular basis for substrate translocation through T3SSs and improve our understanding of bacterial pathogenicity and motility.

In-depth interrogation of protein thermal unfolding data with MoltenProt.

Protein stability is a key factor in successful structural and biochemical research. However, the approaches for systematic comparison of protein stability are limited by sample consumption or compatibility with sample buffer components. Here we describe how miniaturized measurement of intrinsic tryptophan fluorescence (NanoDSF assay) in combination with a simplified description of protein unfolding can be used to interrogate the stability of a protein sample. We demonstrate that improved protein stability measures, such as apparent Gibbs free energy of unfolding, rather than melting temperature Tm , should be used to rank the results of thermostability screens. The assay is compatible with protein samples of any composition, including protein complexes and membrane proteins. Our data analysis software, MoltenProt, provides an easy and robust way to perform characterization of multiple samples. Potential applications of MoltenProt and NanoDSF include buffer and construct optimization for X-ray crystallography and cryo-electron microscopy, screening for small-molecule binding partners and comparison of effects of point mutations.

High-throughput stability screening for detergent-solubilized membrane proteins.

Protein stability in detergent or membrane-like environments is the bottleneck for structural studies on integral membrane proteins (IMP). Irrespective of the method to study the structure of an IMP, detergent solubilization from the membrane is usually the first step in the workflow. Here, we establish a simple, high-throughput screening method to identify optimal detergent conditions for membrane protein stabilization. We apply differential scanning fluorimetry in combination with scattering upon thermal denaturation to study the unfolding of integral membrane proteins. Nine different prokaryotic and eukaryotic membrane proteins were used as test cases to benchmark our detergent screening method. Our results show that it is possible to measure the stability and solubility of IMPs by diluting them from their initial solubilization condition into different detergents. We were able to identify groups of detergents with characteristic stabilization and destabilization effects for selected targets. We further show that fos-choline and PEG family detergents may lead to membrane protein destabilization and unfolding. Finally, we determined thenmodynamic parameters that are important indicators of IMP stability. The described protocol allows the identification of conditions that are suitable for downstream handling of membrane proteins during purification.

An antibody raised against a pathogenic serpin variant induces mutant-like behaviour in the wild-type protein.

A monoclonal antibody (mAb) that binds to a transient intermediate may act as a catalyst for the corresponding reaction; here we show this principle can extend on a macro molecular scale to the induction of mutant-like oligomerization in a wild-type protein. Using the common pathogenic E342K (Z) variant of α1-antitrypsin as antigen-whose native state is susceptible to the formation of a proto-oligomeric intermediate-we have produced a mAb (5E3) that increases the rate of oligomerization of the wild-type (M) variant. Employing ELISA, gel shift, thermal stability and FRET time-course experiments, we show that mAb5E3 does not bind to the native state of α1-antitrypsin, but recognizes a cryptic epitope in the vicinity of the post-helix A loop and strand 4C that is revealed upon transition to the polymerization intermediate, and which persists in the ensuing oligomer. This epitope is not shared by loop-inserted monomeric conformations. We show the increased amenity to polymerization by either the pathogenic E342K mutation or the binding of mAb5E3 occurs without affecting the energetic barrier to polymerization. As mAb5E3 also does not alter the relative stability of the monomer to intermediate, it acts in a manner similar to the E342K mutant, by facilitating the conformational interchange between these two states.