Disulfide Bonds Play a Critical Role in the Structure and Function of the Receptor-binding Domain of the SARS-CoV-2 Spike Antigen.

The current coronavirus pandemic is exerting a tremendously detrimental impact on global health. The Spike proteins of coronaviruses, responsible for cell receptor binding and viral internalization, possess multiple and frequently conserved disulfide bonds raising the question about their role in these proteins. Here, we present a detailed structural and functional investigation of the disulfide bonds of the SARS-CoV-2 Spike receptor-binding domain (RBD). Molecular dynamics simulations of the RBD predict increased flexibility of the surface loops when the four disulfide bonds of the domain are reduced. This flexibility is particularly prominent for the disulfide bond-containing surface loop (residues 456-490) that participates in the formation of the interaction surface with the Spike cell receptor ACE2. In vitro, disulfide bond reducing agents affect the RBD secondary structure, lower its melting temperature from 52 °C to 36-39 °C and decrease its binding affinity to ACE2 by two orders of magnitude at 37 °C. Consistent with these in vitro findings, the reducing agents tris(2-carboxyethyl)phosphine (TCEP) and dithiothreitol (DTT) were able to inhibit viral replication at low millimolar levels in cell-based assays. Our research demonstrates the mechanism by which the disulfide bonds contribute to the molecular structure of the RBD of the Spike protein, allowing the RBD to execute its viral function.

Atomistic structure and dynamics of the human MHC-I peptide-loading complex.

The major histocompatibility complex class-I (MHC-I) peptide-loading complex (PLC) is a cornerstone of the human adaptive immune system, being responsible for processing antigens that allow killer T cells to distinguish between healthy and compromised cells. Based on a recent low-resolution cryo-electron microscopy (cryo-EM) structure of this large membrane-bound protein complex, we report an atomistic model of the PLC and study its conformational dynamics on the multimicrosecond time scale using all-atom molecular dynamics (MD) simulations in an explicit lipid bilayer and water environment (1.6 million atoms in total). The PLC has a layered structure, with two editing modules forming a flexible protein belt surrounding a stable, catalytically active core. Tapasin plays a central role in the PLC, stabilizing the MHC-I binding groove in a conformation reminiscent of antigen-loaded MHC-I. The MHC-I-linked glycan steers a tapasin loop involved in peptide editing toward the binding groove. Tapasin conformational dynamics are also affected by calreticulin through a conformational selection mechanism that facilitates MHC-I recruitment into the complex.

Partial Dissociation of Truncated Peptides Influences the Structural Dynamics of the MHCI Binding Groove.

Antigen processing on MHCI involves the exchange of low-affinity peptides by high-affinity, immunodominant ones. This peptide editing process is mediated by tapasin and ERAAP at the peptide C- and N-terminus, respectively. Since tapasin does not contact the peptide directly, a sensing mechanism involving conformational changes likely allows tapasin to distinguish antigen-loaded MHCI molecules from those occupied by weakly bound, non-specific peptides. To understand this mechanism at the atomic level, we performed molecular dynamics simulations of MHCI allele B*44:02 loaded with peptides truncated or modified at the C- or N-terminus. We show that the deletion of peptide anchor residues leads to reversible, partial dissociation of the peptide from MHCI on the microsecond timescale. Fluctuations in the MHCI α2-1 helix segment, bordering the binding groove and cradled by tapasin in the PLC, are influenced by the peptide C-terminus occupying the nearby F-pocket. Simulations of tapasin complexed with MHCI bound to a low-affinity peptide show that tapasin widens the MHCI binding groove near the peptide C-terminus and weakens the attractive forces between MHCI and the peptide. Our simulations thus provide a detailed, spatially resolved picture of MHCI plasticity, revealing how peptide loading status can affect key structural regions contacting tapasin.

Systematic evaluation of CS-Rosetta for membrane protein structure prediction with sparse NOE restraints.

We critically test and validate the CS-Rosetta methodology for de novo structure prediction of α-helical membrane proteins (MPs) from NMR data, such as chemical shifts and NOE distance restraints. By systematically reducing the number and types of NOE restraints, we focus on determining the regime in which MP structures can be reliably predicted and pinpoint the boundaries of the approach. Five MPs of known structure were used as test systems, phototaxis sensory rhodopsin II (pSRII), a subdomain of pSRII, disulfide binding protein B (DsbB), microsomal prostaglandin E2 synthase-1 (mPGES-1), and translocator protein (TSPO). For pSRII and DsbB, where NMR and X-ray structures are available, resolution-adapted structural recombination (RASREC) CS-Rosetta yields structures that are as close to the X-ray structure as the published NMR structures if all available NMR data are used to guide structure prediction. For mPGES-1 and Bacillus cereus TSPO, where only X-ray crystal structures are available, highly accurate structures are obtained using simulated NMR data. One main advantage of RASREC CS-Rosetta is its robustness with respect to even a drastic reduction of the number of NOEs. Close-to-native structures were obtained with one randomly picked long-range NOEs for every 14, 31, 38, and 8 residues for full-length pSRII, the pSRII subdomain, TSPO, and DsbB, respectively, in addition to using chemical shifts. For mPGES-1, atomically accurate structures could be predicted even from chemical shifts alone. Our results show that atomic level accuracy for helical membrane proteins is achievable with CS-Rosetta using very sparse NOE restraint sets to guide structure prediction. Proteins 2017; 85:812-826. © 2016 Wiley Periodicals, Inc.

Hydration Dynamics of a Peripheral Membrane Protein.

Water dynamics in the hydration shell of the peripheral membrane protein annexin B12 were studied using MD simulations and Overhauser DNP-enhanced NMR. We show that retardation of water motions near phospholipid bilayers is extended by the presence of a membrane-bound protein, up to around 10 Å above that protein. Near the membrane surface, electrostatic interactions with the lipid head groups strongly slow down water dynamics, whereas protein-induced water retardation is weaker and dominates only at distances beyond 10 Å from the membrane surface. The results can be understood from a simple model based on additive contributions from the membrane and the protein to the activation free energy barriers of water diffusion next to the biomolecular surfaces. Furthermore, analysis of the intermolecular vibrations of the water network reveals that retarded water motions near the membrane shift the vibrational modes to higher frequencies, which we used to identify an entropy gradient from the membrane surface toward the bulk water. Our results have implications for processes that take place at lipid membrane surfaces, including molecular recognition, binding, and protein-protein interactions.

Molecular mechanism of peptide editing in the tapasin-MHC I complex.

Immune recognition of infected or malignantly transformed cells relies on antigenic peptides exposed at the cell surface by major histocompatibility complex class I (MHC I) molecules. Selection and loading of peptides onto MHC I is orchestrated by the peptide-loading complex (PLC), a multiprotein assembly whose structure has not yet been resolved. Tapasin, a central component of the PLC, stabilises MHC I and catalyses the exchange of low-affinity against high-affinity, immunodominant peptides. Up to now, the molecular basis of this peptide editing mechanism remained elusive. Here, using all-atom molecular dynamics (MD) simulations, we unravel the atomic details of how tapasin and antigen peptides act on the MHC I binding groove. Force distribution analysis reveals an intriguing molecular tug-of-war mechanism: only high-affinity peptides can exert sufficiently large forces to close the binding groove, thus overcoming the opposite forces exerted by tapasin to open it. Tapasin therefore accelerates the release of low-affinity peptides until a high-affinity antigen binds, promoting subsequent PLC break-down. Fluctuation and entropy analyses show how tapasin chaperones MHC I by stabilising it in a peptide-receptive conformation. Our results explain previous experiments and mark a key step towards a better understanding of adaptive immunity.

Assembly of the MHC I peptide-loading complex determined by a conserved ionic lock-switch.

Salt bridges in lipid bilayers play a decisive role in the dynamic assembly and downstream signaling of the natural killer and T-cell receptors. Here, we describe the identification of an inter-subunit salt bridge in the membrane within yet another key component of the immune system, the peptide-loading complex (PLC). The PLC regulates cell surface presentation of self-antigens and antigenic peptides via molecules of the major histocompatibility complex class I. We demonstrate that a single salt bridge in the membrane between the transporter associated with antigen processing TAP and the MHC I-specific chaperone tapasin is essential for the assembly of the PLC and for efficient MHC I antigen presentation. Molecular modeling and all-atom molecular dynamics simulations suggest an ionic lock-switch mechanism for the binding of TAP to tapasin, in which an unfavorable uncompensated charge in the ER-membrane is prevented through complex formation. Our findings not only deepen the understanding of the interaction network within the PLC, but also provide evidence for a general interaction principle of dynamic multiprotein membrane complexes in immunity.

Mechanistic Basis for Epitope Proofreading in the Peptide-Loading Complex.

The peptide-loading complex plays a pivotal role in Ag processing and is thus central to the efficient immune recognition of virally and malignantly transformed cells. The underlying mechanism by which MHC class I (MHC I) molecules sample immunodominant peptide epitopes, however, remains poorly understood. In this article, we delineate the interaction between tapasin (Tsn) and MHC I molecules. We followed the process of peptide editing in real time after ultra-fast photoconversion to pseudoempty MHC I molecules. Tsn discriminates between MHC I loaded with optimal and MHC I bound to suboptimal cargo. This differential interaction is key to understanding the kinetics of epitope proofreading. To elucidate the underlying mechanism at the atomic level, we modeled the Tsn/MHC I complex using all-atom molecular dynamics simulations. We present a catalytic working cycle, in which Tsn binds to MHC I with suboptimal cargo and thereby adjusts the energy landscape in favor of MHC I complexes with immunodominant epitopes.

An annular lipid belt is essential for allosteric coupling and viral inhibition of the antigen translocation complex TAP (transporter associated with antigen processing).

The transporter associated with antigen processing (TAP) constitutes a focal element in the adaptive immune response against infected or malignantly transformed cells. TAP shuttles proteasomal degradation products into the lumen of the endoplasmic reticulum for loading of major histocompatibility complex (MHC) class I molecules. Here, the heterodimeric TAP complex was purified and reconstituted in nanodiscs in defined stoichiometry. We demonstrate that a single heterodimeric core-TAP complex is active in peptide binding, which is tightly coupled to ATP hydrolysis. Notably, with increasing peptide length, the ATP turnover was gradually decreased, revealing that ATP hydrolysis is coupled to the movement of peptide through the ATP-binding cassette transporter. In addition, all-atom molecular dynamics simulations show that the observed 22 lipids are sufficient to form an annular belt surrounding the TAP complex. This lipid belt is essential for high affinity inhibition by the herpesvirus immune evasin ICP47. In conclusion, nanodiscs are a powerful approach to study the important role of lipids as well as the function, interaction, and modulation of the antigen translocation machinery.

Molecular dynamics of class A ß-lactamases-effects of substrate binding.

The effects of substrate binding on class A ß-lactamase dynamics were studied using molecular dynamics simulations of two model enzymes; 40 100-ns trajectories of the free and substrate-bound forms of TEM-1 (with benzylpenicillin) and PSE-4 (with carbenicillin) were recorded (totaling 4.0 µs). Substrates were parameterized with the CHARMM General Force Field. In both enzymes, the Ω loop exhibits a marked flexibility increase upon substrate binding, supporting the hypothesis of substrate gating. However, specific interactions that are formed or broken in the Ω loop upon binding differ between the two enzymes: dynamics are conserved, but not specific interactions. Substrate binding also has a global structuring effect on TEM-1, but not on PSE-4. Changes in TEM-1's normal modes show long-range effects of substrate binding on enzyme dynamics. Hydrogen bonds observed in the active site are mostly preserved upon substrate binding, and new, transient interactions are also formed. Agreement between NMR relaxation parameters and our theoretical results highlights the dynamic duality of class A ß-lactamases: enzymes that are highly structured on the ps-ns timescale, with important flexibility on the µs-ms timescale in regions such as the Ω loop.

Synergistic applications of MD and NMR for the study of biological systems.

Modern biological sciences are becoming more and more multidisciplinary. At the same time, theoretical and computational approaches gain in reliability and their field of application widens. In this short paper, we discuss recent advances in the areas of solution nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations that were made possible by the combination of both methods, that is, through their synergistic use. We present the main NMR observables and parameters that can be computed from simulations, and how they are used in a variety of complementary applications, including dynamics studies, model-free analysis, force field validation, and structural studies.

Is there nascent structure in the intrinsically disordered region of troponin I?

In striated muscle, the binding of calcium to troponin C (TnC) results in the removal of the C-terminal region of the inhibitory protein troponin I (TnI) from actin. While structural studies of the muscle system have been successful in determining the overall organization of most of the components involved in force generation at the atomic level, the structure and dynamics of the C-terminal region of TnI remains controversial. This domain of TnI is highly flexible, and it has been proposed that this intrinsically disordered region (IDR) regulates contraction via a "fly-casting" mechanism. Different structures have been presented for this region using different methodologies: a single α-helix, a "mobile domain" containing a small ß-sheet, an unstructured region, and a two helix segment. To investigate whether this IDR has in fact any nascent structure, we have constructed a skeletal TnC-TnI chimera that contains the N-domain of TnC (1-90), a short linker (GGAGG), and the C-terminal region of TnI (97-182) and have acquired ¹5N NMR relaxation data for this chimera. We compare the experimental relaxation parameters with those calculated from molecular dynamic simulations using four models based upon the structural studies. Our experimental results suggest that the C-terminal region of TnI does not contain any defined secondary structure, supporting the "fly-casting" mechanism. We interpret the presence of a "plateau" in the ¹5N NMR relaxation data as being an intrinsic property of IDRs. We also identified a more rigid adjacent region of TnI that has implications for muscle performance under ischemic conditions.

TEM-1 backbone dynamics-insights from combined molecular dynamics and nuclear magnetic resonance.

Dynamic properties of class A beta-lactamase TEM-1 are investigated from molecular dynamics (MD) simulations. Comparison of MD-derived order parameters with those obtained from model-free analysis of nuclear magnetic resonance (NMR) relaxation data shows high agreement for N-H moieties within alpha- and beta-secondary structures, but significant deviation for those in loops. This was expected, because motions slower than the protein global tumbling often take place in loop regions. As previously shown using NMR, TEM-1 is a highly ordered protein. Motions are observed within the Omega loop that could, upon substrate binding, stabilize E166 in a catalytically efficient position as the cavity between the protein core and the Omega loop is partially filled. The rigidity of active site residues is consistent with the enzyme high turnover number. MD data are also shown to be useful during the model selection step of model-free analysis: local N-H motions observed over the course of the trajectories help assess whether a peptide plan undergoes low or high amplitude motions on one or more timescales. This joint use of MD and NMR provides a better description of protein dynamics than would be possible using either technique alone.

Inhibition by L-aspartol adenylate of a nondiscriminating aspartyl-tRNA synthetase reveals differences between the interactions of its active site with tRNA(Asp) and tRNA(Asn).

Asparaginyl-tRNA formation in Pseudomonas aeruginosa PAO1 involves a nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) which forms Asp-tRNA(Asp) and Asp-tRNA(Asn), and a tRNA-dependent amidotransferase which transamidates the latter into Asn-tRNA(Asn). We report here that the inhibition of this ND-AspRS by L-aspartol adenylate (Asp-ol-AMP), a stable analog of the natural reaction intermediate L-aspartyl adenylate, is biphasic because the aspartylation of the two tRNA substrates of ND-AspRS, tRNA(Asp) and tRNA(Asn), are inhibited with different Ki values (41 microM and 215 microM, respectively). These results reveal that the two tRNA substrates of ND-AspRS interact differently with its active site. Yeast tRNA(Asp) transcripts with some identity elements replaced by those of tRNA(Asn) have their aspartylation inhibited with Ki values different from that for the wild-type transcript. Therefore, aminoacyl adenylate analogs, which are competitive inhibitors of their cognate aminoacyl-tRNA synthetase, can be used to probe rapidly the role of various structural elements in positioning the tRNA acceptor end in the active site.