Gram-positive pathogenic bacteria Staphylococcus express and secret staphylococcal peroxidase inhibitor (SPIN) proteins to help evade neutrophil-mediated immunity by inhibiting the activity of the main oxidative-defense player myeloperoxidase (MPO) enzyme. SPIN contains a structured 3-helix bundle C-terminal domain, which can specifically bind to MPO with high affinity, and an intrinsically disordered N-terminal domain (NTD), which folds into a structured β-hairpin and inserts itself into the active site of MPO for inhibition. Mechanistic insights of the coupled folding and binding process are needed in order to better understand how residual structures and/or conformational flexibility of NTD contribute to the different strengths of inhibition of SPIN homologs. In this work, we applied atomistic molecular dynamics simulations on two SPIN homologs, from S. aureus and S. delphini , respectively, which share high sequence identity and similarity, to explore the possible mechanistic basis for their different inhibition efficacies on human MPO. Direct simulations of the unfolding and unbinding processes at 450 K reveal that these two SPIN/MPO complexes systems follow surprisingly different mechanisms of coupled binding and folding. While coupled binding and folding of SPIN- aureus NTD is highly cooperative, SPIN- delphini NTD appears to mainly utilize a conformational selection-like mechanism. These observations are in contrast to an overwhelming prevalence of induced folding-like mechanisms for intrinsically disordered proteins that fold into helical structures upon binding. Further simulations of unbound SPIN NTDs at room temperature reveal that SPIN- delphini NTD has a much stronger propensity of forming β-hairpin like structures, consistent with its preference to fold and then bind. These may help explain why the inhibition strength is not well correlated with binding affinity for different SPIN homologs. Altogether, our work establishes the relationship between the residual conformational stability of SPIN-NTD and their inhibitory function, which can help us develop new strategies towards treating Staphylococcal infections.
more »
« less
Computational Ways to Enhance Protein Inhibitor Design
Two new computational approaches are described to aid in the design of new peptide-based drugs by evaluating ensembles of protein structures from their dynamics and through the assessing of structures using empirical contact potential. These approaches build on the concept that conformational variability can aid in the binding process and, for disordered proteins, can even facilitate the binding of more diverse ligands. This latter consideration indicates that such a design process should be less restrictive so that multiple inhibitors might be effective. The example chosen here focuses on proteins/peptides that bind to hemagglutinin (HA) to block the large-scale conformational change for activation. Variability in the conformations is considered from sets of experimental structures, or as an alternative, from their simple computed dynamics; the set of designe peptides/small proteins from the David Baker lab designed to bind to hemagglutinin, is the large set considered and is assessed with the new empirical contact potentials.
more »
« less
- Award ID(s):
- 1661391
- NSF-PAR ID:
- 10288166
- Date Published:
- Journal Name:
- Frontiers in Molecular Biosciences
- Volume:
- 7
- ISSN:
- 2296-889X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
more » « less
Abstract De novo design provides an attractive approach, which allows one to test and refine the principles guiding metalloproteins in defining the geometry and reactivity of their metal ion cofactors. Although impressive progress has been made in designing proteins that bind transition metal ions including iron–sulfur clusters, the design of tetranuclear clusters with oxygen‐rich environments remains in its infancy. In previous work, we described the design of homotetrameric four‐helix bundles that bind tetra‐Zn2+clusters. The crystal structures of the helical proteins were in good agreement with the overall design, and the metal‐binding and conformational properties of the helical bundles in solution were consistent with the crystal structures. However, the correspondingapo ‐proteins were not fully folded in solution. In this work, we design three peptides, based on the crystal structure of the original bundles. One of the peptides forms tetramers in aqueous solution in the absence of metal ions as assessed by CD and NMR. It also binds Zn2+in the intended stoichiometry. These studies strongly suggest that the desired structure has been achieved in theapo state, providing evidence that the peptide is able to actively impart the designed geometry to the metal cluster. -
Longnecker, Richard M. (Ed.)ABSTRACT A cascade of protein-protein interactions between four herpes simplex virus (HSV) glycoproteins (gD, gH/gL, and gB) drive fusion between the HSV envelope and host membrane, thereby allowing for virus entry and infection. Specifically, binding of gD to one of its receptors induces a conformational change that allows gD to bind to the regulatory complex gH/gL, which then activates the fusogen gB, resulting in membrane fusion. Using surface plasmon resonance and a panel of anti-gD monoclonal antibodies (MAbs) that sterically blocked the interaction, we previously showed that gH/gL binds directly to gD at sites distinct from the gD receptor binding site. Here, using an analogous strategy, we first evaluated the ability of a panel of uncharacterized anti-gH/gL MAbs to block binding to gD and/or inhibit fusion. We found that the epitopes of four gD-gH/gL-blocking MAbs were located within flexible regions of the gH N terminus and the gL C terminus, while the fifth was placed around gL residue 77. Taken together, our data localized the gD binding region on gH/gL to a group of gH and gL residues at the membrane distal region of the heterodimer. Surprisingly, a second set of MAbs did not block gD-gH/gL binding but instead stabilized the complex by altering the kinetic binding. However, despite this prolonged gD-gH/gL interaction, “stabilizing” MAbs also inhibited cell-cell fusion, suggesting a unique mechanism by which the fusion process is halted. Our findings support targeting the gD-gH/gL interaction to prevent fusion in both therapeutic and vaccine strategies against HSV. IMPORTANCE Key to developing a human HSV vaccine is an understanding of the virion glycoproteins involved in entry. HSV employs multiple glycoproteins for attachment, receptor interaction, and membrane fusion. Determining how these proteins function was resolved, in part, by structural biology coupled with immunological and biologic evidence. After binding, virion gD interacts with a receptor to activate the regulator gH/gL complex, triggering gB to drive fusion. Multiple questions remain, one being the physical location of each glycoprotein interaction site. Using protective antibodies with known epitopes, we documented the long-sought interaction between gD and gH/gL, detailing the region on gD important to create the gD-gH/gL triplex. Now, we have identified the corresponding gD contact sites on gH/gL. Concurrently we discovered a novel mechanism whereby gH/gL antibodies stabilize the complex and inhibit fusion progression. Our model for the gD-gH/gL triplex provides a new framework for studying fusion, which identifies targets for vaccine development.more » « less
-
more » « less
Abstract Joining biology with materials science requires the ability to design, engineer and control biology/solid‐state materials interfaces at the molecular level. The specific molecular interactions that take place among biomolecules, known as molecular recognition, enable all aspects of molecular processes in living systems prerequisite to the biological functions. Having the ability to establish specific biological interactions between the solid materials and biological constituents is essential for precise design of biologically viable soft interfaces that are molecularly tailored at solid surfaces. Solid‐binding peptides offer excellent opportunities in surface biofunctionalization over the traditionally utilized chemical approaches which generally make use of covalent bonds for surface molecular attachments with limited flexibility. Solid‐binding peptides are selected using directed evolution techniques using genotype to phenotype relationships and therefore referred also as genetically engineered peptides for inorganics (GEPI) and exclusively bind to solid materials using molecular recognition. Here, the peptide has weak interactions at multiple contact points that are established between the biomolecule and the solid lattice, and then folds into a conformation coherent with the underlying solid lattice through self‐organization on the surface. Solid‐binding peptides provide an unprecedented biological advantage as modular building blocks to couple biological and synthetic entities at the bio–solid interfaces. Taking full advantage of biology's versatility, they can easily be engineered to form chimeric molecules with inherent multifunctionality displaying biofunctional molecular entities, such as enzymes, co‐factors, antimicrobial peptides, antibodies, nucleic acids and molecular probes that target biomarkers. This minireview provides an insight into the key principles of solid‐binding peptides for advancing surfaces biofunctionalization by a selected set of examples on chimeric functions built upon linking, displaying and assembling functional molecular moieties at solid surfaces ranging from enzymatic biocatalysis to antimicrobial coatings. Modular multifunctional peptide design offers to tune molecular processes with coupled biological functions for a wide variety of applications in biotechnology, nanotechnology and medicine.
-
more » « less
ABSTRACT Predicting protein conformational changes from unbound structures or even homology models to bound structures remains a critical challenge for protein docking. Here we present a study directly addressing the challenge by reducing the dimensionality and narrowing the range of the corresponding conformational space. The study builds on cNMA—our new framework of partner‐ and contact‐specific normal mode analysis that exploits encounter complexes and considers both intrinsic and induced flexibility. First, we established over a CAPRI (Critical Assessment of PRedicted Interactions) target set that the direction of conformational changes from unbound structures and homology models can be reproduced to a great extent by a small set of cNMA modes. In particular, homology‐to‐bound interface root‐mean‐square deviation (iRMSD) can be reduced by 40% on average with the slowest 30 modes. Second, we developed novel and interpretable features from cNMA and used various machine learning approaches to predict the extent of conformational changes. The models learned from a set of unbound‐to‐bound conformational changes could predict the actual extent of iRMSD with errors around 0.6 Å for unbound proteins in a held‐out benchmark subset, around 0.8 Å for unbound proteins in the CAPRI set, and around 1 Å even for homology models in the CAPRI set. Our results shed new insights into origins of conformational differences between homology models and bound structures and provide new support for the low‐dimensionality of conformational adjustment during protein associations. The results also provide new tools for ensemble generation and conformational sampling in unbound and homology docking. Proteins 2017; 85:544–556. © 2016 Wiley Periodicals, Inc.
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3389/fmolb.2020.607323
