key: cord-0957891-dewbos6u
authors: Costello, Shawn M.; Hobbs, Helen T.; Shoemaker, Sophie R.; Powell, Abigail E.; Lim, Shion A.; Wells, James A.; Kim, Peter S.; Pak, John E.; Marqusee, Susan
title: Mapping Binding Interfaces and Allosteric Changes in the SARS-Cov-2 Spike Protein using Hydrogen/Deuterium Exchange Mass Spectrometry
date: 2021-02-12
journal: Biophysical Journal
DOI: 10.1016/j.bpj.2020.11.980
sha: add8e71e366a63c1d19e04b0a06c59d252f52902
doc_id: 957891
cord_uid: dewbos6u

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School of Pharmacy, Univ Maryland, Baltimore, MD, USA, 2 Department of Chemistry, King's College London, London, United Kingdom, 3 Computational Structural Biology Unit, NINDS NIH, Bethesda, MD, USA. Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) has established itself as a valuable biophysical approach offering meaningful insights into protein structure and dynamics. Recent efforts have aimed to integrate HDX-MS with computational approaches to provide atomistic interpretation of the structural dynamics information garnered. Recently, an HDX-MS based maximum entropy reweighting approach (HDXer) was developed to reweight computationally generated ensembles using HDX-MS data towards high resolution ensemble description of proteins. Here, HDX-MS is used together with enhanced molecular dynamics simulations and HDXer to characterize the structural dynamics of PhuS, a cytoplasmic heme binding protein from P.aeruginosa. Functionally, PhuS shuttles exogenous heme to Heme Oxygenase (HemO) for degradation. Although the crystal structures of unliganded (apo) and heme bound (holo) PhuS are nearly identical, HDX-MS of apo vs holo PhuS revealed large differences in deuterium uptake, notably in C-terminal proximal alpha helices 6, 7 and 8 (a6/7/8). These helices form part of the heme binding pocket and were observed to be mostly labile in apo-PhuS but were largely protected in holo-PhuS. In contrast, the predicted deuterium uptake of a6/7/8 in apo-and holo-PhuS obtained from MD simulations are highly similar to one another and in agreement with the HDX-MS data obtained for holo-PhuS, suggesting that the solution structure of apo-PhuS locally deviates from its crystal structure conformation. The combined use of enhanced sampling MD, HDXer and dimensional reduction reveals an apo-PhuS ensemble in which a6/7/8 are significantly rearranged compared to the crystal structure, including the loss of secondary structure in a6 and the rotation of a7 more than 60 o towards the HemO binding interface. The change in secondary structure was confirmed by circular dichroism spectroscopy of apo and holo-PhuS.

Thermal Transfer from the Surface Loop to the Iron Active Site of Soybean Lipoxygenase Jan Paulo Zaragoza. Department of Chemistry, Univ Calif Berkeley, Berkeley, CA, USA. The rate-limiting chemical reaction catalyzed by soybean lipoxygenase (SLO) involves quantum mechanical tunneling of a hydrogen atom from substrate to its active site ferric-hydroxide cofactor. SLO has emerged as a prototypical system for linking the thermal activation of a protein scaffold to the efficiency of active site chemistry. Significantly, hydrogen-deuterium exchange-mass spectrometry (HDX-MS) experiments on wild type and mutant forms of SLO have uncovered trends in the enthalpic barriers for HDX within a solvent-exposed loop (position 317-334) that correlate well with trends in the corresponding enthalpic barriers for k cat . A model for this behavior posits that collisions between water and loop 317-334 initiate thermal activation at the protein surface that is then propagated 15-34 Å inward toward the reactive carbon of substrate in proximity to the iron catalyst. In this study, we have prepared protein samples containing cysteine residues either at the tip of the loop 317-334 (Q322C) or on a control loop, 586-603 (S596C). Chemical modification of cysteines with the fluorophore 6-bromoacetyl-2-dimethylaminonaphthalene (Badan, BD) provides site-specific probes for the measurement of fluorescence relaxation lifetimes and Stokes shift decays as a function of temperature. While both loops exhibit temperature-independent fluorescence relaxation lifetimes as do the Stokes shifts for S596C-BD, the activation enthalpy for the nanosecond solvent reorganization at Q322C-BD (E a (k solv ) = 2.8(0.9) kcal/mol)) approximates the enthalpy of activation for catalytic C-H activation (E a (k cat ) = 2.1(0.2) kcal/ mol). This study establishes and validates methodology for measuring rates of rapid local motions at the protein/solvent interface of SLO. These new findings, when combined with previously published correlations between protein motions and the rate limiting hydride transfer in a thermophilic alcohol dehydrogenase, provide experimental evidence for thermally-induced 'protein quakes' as the origin of enthalpic barriers in catalysis.

Allosteric Regulation of the Activity of BY-Kinases, a Unique Family of Bacterial Protein Tyrosine Kinases Fatlum Hajredini 1,2 , Andrea Piserchio 2 , Rinat Abzalimov 3 , Ranajeet Ghose 2,4 . 1 Graduate Program in Biochemistry, the Graduate Center of CUNY, New York, NY, USA, 2 Department of Chemistry and Biochemistry, the City University of New York, New York, NY, USA, 3 CUNY Advanced Science Research Center, the Graduate Center of CUNY, New York, NY, USA, 4 Graduate Programs in Biochemistry, Chemistry, and Physics, the Graduate Center of CUNY, New York, NY, USA. BY-kinases comprise a family of protein tyrosine kinases that are highly conserved in both Gram-positive and Gram-negative bacteria but are without any eukaryotic orthologs. BY-kinases are involved in a variety of physiological processes, most notably in the production of polysaccharides involved in capsule synthesis or biofilm formation. BY-kinases are unique in that their catalytic domains are closely related to the P-loop ATPases and do not encode the dual-lobed architecture characteristic of eukaryotic protein kinases. BYkinases are proposed to function through an assembly/disassembly process regulated by the phosphorylation state of a cluster of tyrosine residues found on the C-terminal tail of their catalytic domains. Using the catalytic domain of a prototypical BY-kinase, Escherichia. coli Wzc and utilizing enhanced sampling molecular dynamics, solution NMR and complimentary biophysical approaches, we demonstrate the presence of a long-range allosteric network that connects the structural elements that facilitate the assembly (oligomerization) process to the kinase active site. This network regulates the conformational states of key catalytic elements and thereby influences nucleotide exchange. We further demonstrate that a single, highly conserved active site residue represents a key node within this allosteric network. Our results highlight a novel regulatory mechanism that enables the BY-kinases to transfer the g-phosphate of ATP to a tyrosine -OH moiety, rather than one on water, while deploying an ATPase fold. University of California San Francisco, San Francisco, CA, USA, 6 Chan Zuckerberg Biohub, San Francisco, CA, USA. Coronaviruses (CoVs), including the severe acute respiratory syndrome (SARS) and middle east respiratory syndrome (MERS) viruses, have and continue to pose a major threat to human health. In October of 2020 the COVID-19 pandemic, caused by the coronavirus SARS-CoV-2, surpassed one million global deaths. CoVs are enveloped positive strand RNA viruses that display surface spike proteins which recognize host receptors. The spike proteins then undergo conformational changes that allow for attachment to the host membrane and eventually facilitate membrane fusion and viral entry. Due to their exposure on the virus surface and their essential role in coronavirus infection, most antibody development strategies and many therapeutic development strategies have focused on the spike protein.

While much focus has been given to the structure of spike, successful design of therapeutics requires understanding the conformational dynamics and alternative conformations not accessible using traditional structural methods. We have successfully applied Hydrogen/Deuterium Exchange Mass Spectrometry (HDX/MS) on this large (>400 kDa) glycosylated trimeric complex to investigate these important conformational changes; allowing us to identify the binding interfaces as well as the induced allosteric changes upon binding to the human receptor ACE2, neutralizing patient antibodies, and synthetic binders. We also compare the conformational flexibility of coronavirus homologues and naturally occurring SARS-CoV-2 spike variants allowing us to connect changes in the conformational ensemble to functional, phenotypic differences in these variants. Understanding the native conformational ensemble, and interactions between receptors or antibodies with CoV spike proteins will not only improve our understanding of CoV biology and the host immune response to CoV but also aid in the design of therapeutics and vaccines for current and future CoVs.

Wei Chen, Elizabeth A. Komives. Chemistry and Biochemistry, Univ Calif San Diego, La Jolla, CA, USA. The NF-kB family of transcription factor is a central mediator of immune and imflammatory responses. NF-kB functions as a dimer and recognize DNA through the two N-terminal domains (NTDs). Previous hydrogen-deuterium exchange mass spectrometry, stopped-flow experiments and molecular dynamics simulations suggested the two NTDs can undergo relative motions which plays an important role in the kinetics of DNA association and dissociation. Here we showed by single-molecule FRET the direct evidence of relative domain motions in the NF-kB RelA-p50 dimer. Surprisingly, the two NTDs underwent slow heterogeneous motions on the timescale of seconds to minutes. These motions are scaled down when NF-kB is bound to DNA and allosterically altered by the inhibitor protein IkBa, which is known to accelerate DNA dissociation and prevent DNA binding. A new DNA-bound conformation unexpected from crystal structure was also observed. Together, our results provide a direct visualization and quantitative characterization of large-scale domain motions of NF-kB and shed light on the understanding of the role of protein dynamics in protein-protein interactions.

Insight Into Cullin Ring Ligase and Substrate Binding Philip R. Belzeski 1 , Ryan Lumpkin 1 , Christopher Condon 2 , Elizabeth A. Komives 1 . 1 Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA, 2 Salk Institute, La Jolla, CA, USA. Ubiquitylation helps regulate protein degradation and maintain normal cellular function. E3 ubiquitin ligases facilitate the attachment of ubiquitin molecules to substrate proteins. The most common class of E3 ubiquitin ligase is the Cullin RING Ligase (CRL), which is a complex of proteins containing a Cullin protein as a scaffold. Cullin 5 associates with one of 18 Ankyrin-Repeat and SOCS Box (ASB) proteins, and the RING-box (RXB) protein, RBX2 which binds the ubiquitylated E2. When substrate binds, the ubiquitin is transferred from the E2 protein to an exposed lysine residue on the substrate. I am exploring the ubiquitylation of putative substrates identified in a proteomics pull-down study of the ASB proteins. I have assessed several putative substrates for ubiquitylation by the ASB CRL and have identified additional substrates. I have used size exclusion chromatography to assess whether the substrate binds with high affinity to the ASB CRL. Additionally, I have tested whether Nedd8 is necessary to activate the CRL to achieve ubiquitylation. Finally, I will present HDX-MS data to identify the interface between the ligase and the substrate.

Disulfide Reduction Allosterically Destabilizes the b-Ladder Sub-Domain Assembly Within the NS1 Dimer of ZIKV Priti Roy 1 , Subhajit Roy 2 , Neelanjana Sengupta 3 . 1 Biological Sciences, IISER Kolkata, Kalyani, India, 2 Centre for Excellence in Basic SCIENCES (CBS), Vidyanagari, India, 3 Biological Sciences, IISER Kolkata, Mohanpur, India. The Zika virus (ZIKV) was responsible for a recent debilitating epidemic that till date has no cure. A potential way to reduce ZIKV virulence is to limit the action of the non-structural proteins involved in its viral replication. One such protein, NS1, encoded as a monomer by the viral genome, plays a major role via symmetric oligomerization. We examine the homodimeric structure of the dominant b-ladder segment of NS1 with extensive all atom molecular dynamics. We find it stably bounded by two spatially separated interaction clusters (C1 and C2) with significant differences in the nature of their interactions. Four pairs of distal, intra-monomeric disulfide bonds are found to be coupled to the stability, local structure, and wettability of the interfacial region. Symmetric reduction of the intra-monomeric disulfides triggers marked dynamical heterogeneity, interfacial wettability and asymmetric salt bridging propensity. Harnessing the model-free Lipari-Szabo based formalism for estimation of conformational entropy (S conf ), we find clear signatures of heterogeneity in the monomeric conformational entropies. The observed asymmetry, very small in the unperturbed state, expands significantly in the reduced states. This allosteric effect is most noticeable in the electrostatically bound C2 cluster that underlies the greatest stability in the unperturbed state. Allosteric induction of conformational and thermodynamic asymmetry is expected to affect the pathways leading to symmetric higher ordered oligomerization, and thereby affect crucial replication pathways.

Their dynamic conformational ensembles encompassing various inactive and active states can be biased by the lipids and surrounding aqueous environments. By using different polyethylene glycol (PEG) solutions, we explored the effect of osmotic pressure and lipid bilayer composition on the metarhodopsin equilibrium of the archetypical GPCR rhodopsin in native membranes and POPC recombinant membranes. Our results show a flood of 80 water molecules into the rhodopsin interior during photoactivation

Dehydrating conditions favor Meta-I through an efflux of water from the protein interior, while increasing bilayer thickness and the monolayer spontaneous curvature favor Meta-II. The osmotic effect on the protein is more significant than the effect of the lipid bilayer. However, small osmolytes favored the Meta-II state at lower concentrations, because they can penetrate the protein core giving a lower excluded volume, decreasing the osmotic effect on the protein and favoring the Meta-II state. Furthermore, the metarhodopsin equilibrium was shifted towards the Meta-I state in POPC recombinant membranes compared to the native membrane environment. Analysis of transducin C-terminal peptide-binding isotherms revealed that the binding affinity is significantly decreased when the lipid environment is changed from the native lipids to POPC lipids. The POPC lipid membrane has zero-spontaneous curvature that shifts the equilibrium towards the more compact, inactive Meta-I state. By contrast, the native lipid membrane environment has a negative spontaneous curvature that favors the more expanded state of Meta-II

621-Pos Calcium Dissociation of HCM Causative R21C Troponin I Mutation Romi Castillo 1 , Anthony Baldo 2