12 Jul 2021

The Contribution of Biophysics and Structural Biology to Current Advances in COVID-19 (Annual reviews of Biophysics)

Critical to viral infection are the multiple interactions between viral proteins and host-cell counterparts. The first such interaction is the recognition of viral envelope proteins by surface receptors that normally fulfil other physiological roles, a hijacking mechanism perfected over the course of evolution. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), has successfully adopted this strategy using its spike glycoprotein to dock on the membrane-bound metalloprotease angiotensin-converting enzyme 2 (ACE2). The crystal structures of several SARS-CoV-2 proteins alone or in complex with their receptors or other ligands were recently solved at an unprecedented pace. This accomplishment is partly due to the increasing availability of data on other coronaviruses and ACE2 over the past 18 years. Likewise, other key intervening actors and mechanisms of viral infection were elucidated with the aid of biophysical approaches. An understanding of the various structurally important motifs of the interacting partners provides key mechanistic information for the development of structure-based designer drugs able to inhibit various steps of the infective cycle, including neutralizing antibodies, small organic drugs, and vaccines. This review analyzes current progress and the outlook for future structural studies.

18 May 2021

Lead compounds for the development of SARS-CoV-2 3CL protease inhibitors (Nature Communications)

Stockwell, B.R., Chavez, A., and Ho, D.D. report the identification of three structurally diverse compounds – compound 4, GC376, and MAC-5576 – as inhibitors of the SARS-CoV-2 3CL protease. Structures of each of these compounds in complex with the protease revealed strategies for further development, as well as general principles for designing SARS-CoV-2 3CL protease inhibitors. These compounds may therefore serve as leads for the basis of building effective SARS-CoV-2 3CL protease inhibitors.

18 May 2021

The antigenic anatomy of SARS-CoV-2 receptor binding domain (Cell)

Antibodies are crucial to immune protection against SARS-CoV-2, with some in emergency use as therapeutics. Here, we identify 377 human monoclonal antibodies (mAbs) recognizing the virus spike and focus mainly on 80 that bind the receptor binding domain (RBD). We devise a competition data-driven method to map RBD binding sites. We find that although antibody binding sites are widely dispersed, neutralizing antibody binding is focused, with nearly all highly inhibitory mAbs (IC50 < 0.1 mu g/mL) blocking receptor interaction, except for one that binds a unique epitope in the N-terminal domain. Many of these neutralizing mAbs use public V-genes and are close to germline. We dissect the structural basis of recognition for this large panel of antibodies through X-ray crystallography and cryoelectron microscopy of 19 Fab-antigen structures. We find novel binding modes for some potently inhibitory antibodies and demonstrate that strongly neutralizing mAbs protect, prophylactically or therapeutically, in animal models.

11 May 2022

True-atomic-resolution insights into the structure and functional role of linear chains and low-barrier hydrogen bonds in proteins (Nature Structural & Molecular Biology)

Hydrogen bonds are fundamental to the structure and function of biological macromolecules and have been explored in detail. The chains of hydrogen bonds (CHBs) and low-barrier hydrogen bonds (LBHBs) were proposed to play essential roles in enzyme catalysis and proton transport. However, high-resolution structural data from CHBs and LBHBs is limited. The challenge is that their ‘visualization’ requires ultrahigh-resolution structures of the ground and functionally important intermediate states to identify proton translocation events and perform their structural assignment. Ttrue-atomic-resolution structures of the light-driven proton pump bacteriorhodopsin, a model in studies of proton transport, show that CHBs and LBHBs not only serve as proton pathways, but also are indispensable for long-range communications, signaling and proton storage in proteins. The complete picture of CHBs and LBHBs discloses their multifunctional roles in providing protein functions and presents a consistent picture of proton transport and storage resolving long-standing debates and controversies.

11 May 2022

What's next for AlphaFold and the AI protein-folding revolution (Nature)

DeepMind software that can predict the 3D shape of proteins is already changing biology. This News Feature published in Nature lists several examples of successful deployment of AlphaFold and RoseTTAFold software and comments their performance.

6 Apr 2022

Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase alpha-aminoacrylate intermediate (PNAS)

NMR-assisted crystallography—the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry—holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5′-phosphate–dependent enzymes that catalyze β-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate β-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid–base catalytic residue βLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate C^β and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.

6 Apr 2022

Structure and mechanism of a methyltransferase ribozyme (Nature Chemical Biology)

Known ribozymes in contemporary biology perform a limited range of chemical catalysis, but in vitro selection has generated species that catalyze a broader range of chemistry; yet, there have been few structural and mechanistic studies of selected ribozymes. A ribozyme has recently been selected that can catalyze a site-specific methyl transfer reaction. The authors have solved the crystal structure of this ribozyme at a resolution of 2.3 Å, showing how the RNA folds to generate a very specific binding site for the methyl donor substrate. The structure immediately suggests a catalytic mechanism involving a combination of proximity and orientation and nucleobase-mediated general acid catalysis. The mechanism is supported by the pH dependence of the rate of catalysis. A selected methyltransferase ribozyme can thus use a relatively sophisticated catalytic mechanism, broadening the range of known RNA-catalyzed chemistry.

6 Apr 2022

Structural basis for mismatch surveillance by CRISPR–Cas9 (Nature)

CRISPR–Cas9 as a programmable genome editing tool is hindered by off-target DNA cleavage, and the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood. Although Cas9 variants with greater discrimination against mismatches have been designed, these suffer from substantially reduced rates of on-target DNA cleavage. Here the authors used kinetics-guided cryo-electron microscopy to determine the structure of Cas9 at different stages of mismatch cleavage. Theyobserved a distinct, linear conformation of the guide RNA–DNA duplex formed in the presence of mismatches, which prevents Cas9 activation. Although the canonical kinked guide RNA–DNA duplex conformation facilitates DNA cleavage, they observe that substrates that contain mismatches distal to the protospacer adjacent motif are stabilized by reorganization of a loop in the RuvC domain. Mutagenesis of mismatch-stabilizing residues reduces off-target DNA cleavage but maintains rapid on-target DNA cleavage. By targeting regions that are exclusively involved in mismatch tolerance, they provide a proof of concept for the design of next-generation high-fidelity Cas9 variants.

6 Apr 2022

Structural basis of human telomerase recruitment by TPP1-POT1 (Science)

Telomerase maintains genome stability by extending the 3′ telomeric repeats at eukaryotic chromosome ends, thereby counterbalancing progressive loss caused by incomplete genome replication. In mammals, telomerase recruitment to telomeres is mediated by TPP1, which assembles as a heterodimer with POT1. The authors report cryo–electron microscopy

structures of DNA-bound telomerase in complex with TPP1 and with TPP1-POT1 at 3.2- and 3.9-angstrom resolution, respectively. Their structures define interactions between telomerase and TPP1-POT1 that are crucial for telomerase recruitment to telomeres. The presence of TPP1-POT1 stabilizes the DNA, revealing an unexpected path by which DNA exits the telomerase active site and a DNA anchor site on telomerase that is important for telomerase processivity. Their findings rationalize extensive prior genetic and biochemical findings and provide a framework for future mechanistic work on telomerase regulation.

6 Apr 2022

Structural basis for activation and gating of IP3receptors (Nature Communications)

A pivotal component of the calcium (Ca²⁺) signaling toolbox in cells is the inositol 1,4,5-triphosphate (IP₃) receptor (IP₃R), which mediates Ca²⁺ release from the endoplasmic reticulum (ER), controlling cytoplasmic and organellar Ca²⁺ concentrations. IP₃Rs are co-activated by IP₃ and Ca²⁺, inhibited by Ca²⁺ at high concentrations, and potentiated by ATP. However, the underlying molecular mechanisms are unclear. Here Erkan Karakas et. al. report cryo-electron microscopy (cryo-EM) structures of human type-3 IP₃R obtained from a single dataset in multiple gating conformations: IP₃-ATP bound pre-active states with closed channels, IP₃-ATP-Ca²⁺ bound active state with an open channel, and IP₃-ATP-Ca²⁺ bound inactive state with a closed channel.The structures demonstrate how IP₃-induced conformational changes prime the receptor for activation by Ca²⁺, how Ca²⁺ binding leads to channel opening, and how ATP modulates the activity, providing insights into the long-sought questions regarding the molecular mechanism underpinning receptor activation and gating.

6 Apr 2022

Structure determination of high-energy states in a dynamic protein ensemble (Nature)

Macromolecular function frequently requires that proteins change conformation into high-energy states. However, methods for solving the structures of these functionally essential, lowly populated states are lacking. Here Dorothee Kern at.al. develop a method for high-resolution structure determination of minorly populated states by coupling NMR spectroscopy-derived pseudocontact shifts (PCSs) with Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion (PCS–CPMG). Their approach additionally defines the corresponding kinetics and thermodynamics of high-energy excursions, thereby characterizing the entire free-energy landscape. Using a large set of simulated data for adenylate kinase (Adk), calmodulin and Src kinase, they find that high-energy PCSs accurately determine high-energy structures (with a root mean squared deviation of less than 3.5 angström). Applying their methodology to Adk during catalysis, they find that the high-energy excursion involves surprisingly small openings of the AMP and ATP lids. This previously unresolved high-energy structure solves a longstanding controversy about conformational interconversions that are rate-limiting for catalysis. Primed for either substrate binding or product release, the high-energy structure of Adk suggests a two-step mechanism combining conformational selection to this state, followed by an induced-fit step into a fully closed state for catalysis of the phosphoryl-transfer reaction. Unlike other methods for resolving high-energy states, such as cryo-electron microscopy and X-ray crystallography, their solution PCS–CPMG approach excels in cases involving domain rearrangements of smaller systems (less than 60 kDa) and populations as low as 0.5%, and enables the simultaneous determination of protein structure, kinetics and thermodynamics while proteins perform their function.