Research

Macromolecular machines occur ubiquitously in nature where they achieve a broad spectrum of biological functions sustaining life. They are made of proteins which often accomplish their tasks as part of a multi-subunit complex, either stable or transient. These nano-assemblies are of significant medical interest since perturbations of protein/protein, protein/carbohydrate or protein/nucleic acid interactions can lead to a number of diseases. A deep understanding of these molecular machines is therefore key to understanding biological processes and expediting advances in medicine.

Our main research interest is to decipher the structure and function of macromolecular complexes involved in the pathogenesis of infectious diseases to provide avenues for creating vaccines and therapeutics. We use a multi-disciplinary approach involving cryo-electron microscopy, X-ray crystallography and mass spectrometry complemented by various biochemical and biophysical techniques to obtain multi-scale data ranging from atom to whole-cell.

 

Glycan shield and fusion activation of a deltacoronavirus spike glycoprotein fine-tuned for enteric infections.
Coronaviruses recently emerged as major human pathogens causing outbreaks of severe acute respiratory syndrome and Middle-East respiratory syndrome. They utilize the spike (S) glycoprotein anchored in the viral envelope to mediate host attachment and fusion of the viral and cellular membranes to initiate infection. The S protein is a major determinant of the zoonotic potential of coronaviruses and is also the main target of the host humoral immune response. We report here the 3.5 Å resolution cryo-electron microscopy structure of the S glycoprotein trimer from the pathogenic porcine deltacoronavirus (PDCoV), which belongs to the recently identified delta genus. Structural and glycoproteomics data indicate that the glycans of PDCoV S are topologically conserved when compared with the human respiratory coronavirus HCoV-NL63 S, resulting in similar surface areas being shielded from neutralizing antibodies and implying that both viruses are under comparable immune pressure in their respective hosts. The structure further reveals a shortened S2’ activation loop, containing a reduced number of basic amino acids, which participates to rendering the spike largely protease-resistant. This property distinguishes PDCoV S from recently characterized betacoronavirus S proteins and suggests that the S protein of enterotropic PDCoV has evolved to tolerate the protease-rich environment of the small intestine and to fine-tune its fusion activation to avoid premature triggering and reduction of infectivity.
Reference:
Xiong X, Tortorici MA, Snijder J, Yoshioka C, Walls AC, Li W, McGuire AT, Rey FA, Bosch BJ and Veesler D. Glycan shield and fusion activation of a deltacoronavirus spike glycoprotein fine-tuned for enteric infections. J Virol (in press). doi:10.1128/JVI.01628-17

The therapeutic antibody LM609 selectively inhibits ligand binding to human αVβ3 integrin via steric hindrance.
The LM609 antibody specifically recognizes αVβ3 integrin and inhibits angiogenesis, bone resorption, and viral infections in an arginine-glycine-aspartate independent manner. LM609 entered phase II clinical trials for the treatment of several cancers and was also used for αVβ3-targeted radio-immunotherapy. To elucidate the mechanisms of recognition and inhibition of αVβ3 integrin, we solved the structure of the LM609 antigen-binding fragment by X-ray crystallography and determined its binding affinity for αVβ3. Using single-particle electron microscopy we show that LM609 binds at the interface between the β-propeller domain of the αV chain and the βI domain of the β3 chain, near the RGD-binding site, of all observed integrin conformational states. Integrating this data with fluorescence size-exclusion chromatography, we demonstrate that LM609 sterically hinders access of large ligands to the RGD binding pocket, without obstructing it. This work provides a structural framework to expedite future efforts utilizing LM609 as a diagnostic or therapeutic tool.
Reference: Borst AJ, James ZM, Zagotta WN, Ginsberg M, Rey FA, DiMaio F, Backovic M and Veesler D. The therapeutic antibody LM609 selectively inhibits ligand binding to human αvβ3 integrin via steric hindrance. Structure (2017) 25(11):1732-1739.

Tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion.
The tremendous pandemic potential of coronaviruses was demonstrated twice in the past few decades by two global outbreaks of deadly pneumonia. The coronavirus spike (S) glycoprotein initiates infection, by promoting fusion of the viral and cellular membranes, through conformational changes that remain largely uncharacterized. Here we report the cryo-electron microscopy structure of a coronavirus S glycoprotein in the post-fusion state, showing large-scale secondary, tertiary and quaternary rearrangements compared to the pre-fusion trimer, and rationalizing the free energy landscape of this conformational machine. We also biochemically characterized the molecular events associated with refolding of the metastable pre-fusion S glycoprotein to the post-fusion conformation using limited proteolysis, mass spectrometry and single particle electron microscopy. The observed similarity between post-fusion coronavirus S and paramyxovirus F structures demonstrates that a conserved refolding trajectory mediates entry of these viruses and supports the evolutionary relatedness of their fusion subunits. Finally, our data provides a structural framework for understanding the mode of neutralization of antibodies targeting the fusion machinery and for engineering next-generation subunit vaccines or inhibitors against this medically important virus family.
Reference: Walls AC, Tortorici MA, Snijder J, Xiong X, Bosch BJ, Rey FA and Veesler D. Tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion. Proc Natl Acad Sci. (2017) 114(42):11157-62.

CryoEM Structure of a Prokaryotic Cyclic Nucleotide-Gated Ion Channel.
Cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels play crucial physiological roles in phototransduction, olfaction, and cardiac pace making. These channels are characterized by the presence of a carboxy-terminal cyclic nucleotide-binding domain (CNBD) that connects to the channel pore via a C-linker domain. Although cyclic nucleotide binding has been shown to promote CNG and HCN channel opening, the precise mechanism underlying gating remains poorly understood. Here we used cryo-electron microscopy (cryoEM) to determine the structure of the intact LliK cyclic nucleotide-gated channel isolated from Leptospira licerasiae – which shares sequence similarity to eukaryotic CNG and HCN channels – in the presence of a saturating concentration of cyclic AMP. A short S4-S5 linker connects nearby voltage-sensing and pore domains to produce a non-domain-swapped transmembrane architecture, which appears to be a hallmark of this channel family. We also observe major conformational changes of the LliK C-linkers and CNBDs relative to the crystal structures of isolated C-linker/CNBD fragments and the cryoEM structures of related CNG, HCN, and KCNH channels. The novel conformation of our LliK structure may represent a functional state of this channel family not captured in previous studies.
Reference: James ZM, Borst AJ, Haitin Y, Frenz B, DiMaio F, Zagotta WN and Veesler D. CryoEM structure of a prokaryotic cyclic nucleotide-gated ion channel. Proc Natl Acad Sci. (2017) 114(17):4430-35.

Glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy.
The threat of a major coronavirus pandemic urges the development of suitable strategies to combat these pathogens. HCoV-NL63 is an -coronavirus that can cause severe lower respiratory tract infections requiring hospitalization. We report here the 3.4 Å resolution cryo-electron microscopy reconstruction of the HCoV-NL63 coronavirus spike glycoprotein trimer, which is the conformational machine responsible for entry into host cells and the sole target of neutralizing antibodies during infection. The map resolves the extensive glycan shield obstructing the protein surface and, in combination with mass-spectrometry, provides a structural framework to understand accessibility to antibodies. The structure also reveals a remarkable modular architecture of the receptor-binding subunit and the complete architecture of the fusion machinery including the triggering loop and the C-terminal domains, which contribute to anchoring the trimer to the viral membrane. Our data further suggest that HCoV-NL63 and other coronaviruses use molecular trickery, based on masking of epitopes with glycans and activating conformational changes, to evade the immune system of infected hosts.
Reference: Walls AC, Tortorici MA, Frenz B, Snijder J, Rey FA, DiMaio F, Bosch BJ and Veesler D. Glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy. Nat Struct Mol Biol. (2016) 23(10):899-905.

 

Cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer.
The tremendous pandemic potential of coronaviruses was demonstrated twice in the last decades by two global outbreaks of deadly pneumonia. Entry of coronaviruses into cells is mediated by the transmembrane spike glycoprotein S, which forms a trimer carrying receptor-binding and membrane fusion functions1. S also contains the principal antigenic determinants and is the target of neutralizing antibodies. Here we present the structure of a murine coronavirus S trimer ectodomain determined at 4.0 Å resolution by single particle cryo-electron microscopy. It reveals the metastable pre-fusion architecture of S and highlights key interactions stabilizing it. The structure shares a common core with paramyxovirus F proteins2,3, implicating mechanistic similarities and an evolutionary connection between these viral fusion proteins. The accessibility of the highly conserved fusion peptide at the periphery of the trimer indicates potential vaccinology strategies to elicit broadly neutralizing antibodies against coronaviruses. Finally, comparison with crystal structures of human coronavirus S domains allows rationalization of the molecular basis for species specificity based on the use of spatially contiguous but distinct domains.
Reference: Walls AC, Tortorici MA, Bosch BJ, Frenz B, Rottier PJM, DiMaio F, Rey FA and Veesler D. Cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer. Nature. (2016). 531:114-117.

Thermoplasma acidophilum 20S proteasome. Reference: Campbell MG, Veesler D, Cheng A; Potter CS and Carragher B. 2.8 Å resolution reconstruction of the Thermoplasma acidophilum 20 S proteasome using cryo-electron microscopy. eLife 2015;10.7554/eLife.06380

2.8 Å resolution reconstruction of the Thermoplasma acidophilum 20 S proteasome using cryo-electron microscopy.
Recent developments in detector hardware and image-processing software have revolutionized single particle cryo-electron microscopy (cryoEM) and led to a wave of near-atomic resolution (typically ∼3.3 ° A) reconstructions. Reaching resolutions higher than 3 °A is a prerequisite for structure-based drug design and for cryoEM to become widely interesting to pharmaceutical industries. We report here the structure of the 700 kDa Thermoplasma acidophilum 20S proteasome (T20S), determined at 2.8 °A resolution by single-particle cryoEM. The quality of the reconstruction enables identifying the rotameric conformation adopted by some amino-acid side chains (rotamers) and resolving ordered water molecules, in agreement with the expectations for crystal structures at similar resolutions. The results described in this manuscript demonstrate that single particle cryoEM is capable of competing with X-ray crystallography for determination of protein structures of suitable quality for rational drug design.
Reference: Campbell MG, Veesler D, Cheng A, Potter CS and Carragher B. 2.8 Å resolution reconstruction of the Thermoplasma acidophilum 20 S proteasome using cryo-electron microscopy. eLife 2015;10.7554/eLife.06380