Structural Biology of Infectious Diseases

Conference: 2021: 71st ACA Annual Meeting
08/03/2021: 12:00 PM - 3:00 PM
4.1.5 
Oral Session 

Description

This session will focus on the structural basis of host–pathogen interactions and the application of structural biology to combat infectious diseases. Possible topics include, but are not limited to, structural studies on viral, bacterial, and fungal proteins and their mechanisms of action or interactions with host cell molecules, receptors, and antibodies. Structure-based vaccine and antibody development is also of interest.

Presentations

Recognition of the Plasmodium spp. circumsporozoite protein by malaria inhibitory antibodies

Sporozoites of the malaria parasites from Plasmodium genus express a surface circumsporozoite protein (CSP) that contains an unusual central region consisting of multiple amino acids repeats. The central region of CSP is immunodominant and antibodies targeting the repeats can inhibit sporozoite infectivity. CSP is the antigenic component of the most advanced malaria vaccine to date - RTS,S/AS01. As RTS,S/AS01 offers only modest protection, it is important to gain a better molecular understanding of the humoral immune response against this Plasmodium antigen to guide the design of superior vaccines.
In this study, we have analyzed the structure and molecular interactions of potent monoclonal antibodies (mAbs) using molecular dynamics simulations, X-ray crystallography, and cryoEM. We discovered that inhibitory mAbs can accommodate subtle variances of the CSP repeating motifs, and, upon binding, induce structural ordering of CSP through homotypic interactions. Interestingly, some mAb/CSP complexes formed spiral assemblies with a varied degree of compactness. We propose that homotypic antibody interactions leading to antibody clustering around CSP is a general immune mechanism of mammals to interact with repeating motifs on sporozoites across Plasmodium species. 

View Abstract 668

Author

Iga Kucharska, The Peter Gilgan Centre for Research and Learning, Hospital for Sick Children Toronto

Additional Author(s)

Elaine Thai, The Peter Gilgan Centre for Research and Learning, Hospital for Sick Children Toronto
Ananya Srivastava, The Peter Gilgan Centre for Research and Learning, Hospital for Sick Children Toronto
John Rubinstein, The Hospital for Sick Children Toronto, Ontario 
Régis Pomès, The Peter Gilgan Centre for Research and Learning, Hospital for Sick Children Toronto
Jean-Philippe Julien, The Peter Gilgan Centre for Research and Learning, Hospital for Sick Children Toronto

Structurally investigating a niche pathway for chemical reversal of proline hydroxylation in the pathogen C. difficile

The glycyl radical enzyme (GRE) family utilizes a glycyl radical cofactor, installed by AdoMet radical activating enzymes, to catalyze difficult chemical reactions in a variety of microbial metabolic pathways. Although glycyl radical enzymes are widely encoded and expressed by bacteria found in the gut microbiome, these enzymes remain largely uncharacterized. Recently, a new glycyl radical enzyme was discovered to catalyze the dehydration of trans-4-hydroxy-L-proline (4-Hyp) to 1-pyrroline-5-carboxylic acid. Bioinformatics studies by the Balskus lab show that this hydroxyproline dehydratase (HypD) is the second most prominent GRE in the human gut microbiome and is encoded by 360 bacterial genomes, including the human pathogen C. difficile. HypD presents a pathway for bacteria to reverse 4-Hyp post-translational modifications, the most common post-translational modification in animals which was previously thought to be irreversible. Furthermore, the bacteria that encode HypD are known to use 4-Hyp as an electron acceptor during amino acid fermentation, their primary method of generating adenosine triphosphate (ATP). However, the enzyme responsible for assimilating 4-Hyp into this pathway has remained unknown until now. HypD could be the missing puzzle piece to understanding how these bacteria use the abundant metabolite 4-Hyp in energy production, while also symbiotically providing humans with a method for recycling this common amino acid. In order to elucidate the mechanism for how HypD performs the dehydration of hydroxyproline, we aimed to characterize HypD from C. difficile, in the presence of its substrate. Here, we have solved a 2.05-Å resolution structure for HypD by molecular replacement. Subsequently, a structure for HypD with its substrate 4-Hyp bound in the active site was solved to 2.52-Å resolution. These structures and accompanying biochemical studies have led us to identify key catalytic residues and have provided insight into the mechanism for 4-Hyp dehydration. 

View Abstract 475

Author

Lindsey Backman, Massachusetts Institute of Technology Cambridge, MA 

Structures of Usutu SAAR-1776 virus: a comparison with known structures of mature flaviviruses

Flaviviruses are arthropod-borne viruses that are most commonly transmitted to humans via mosquitoes or ticks. These viruses are notorious for their infrequent, sudden outbreaks in the human population, which involve severe disease manifestations such as encephalitis or hemorrhagic fever; yet, the details of immunopathogenesis of the viruses and their interplay are not fully understood. Usutu virus (USUV), a flavivirus belonging to the Japanese encephalitis serocomplex and serologically closely related to the West Nile virus, is rampant in Europe and increasingly being recognized for its ability to cause severe neurological complications in humans. However, the prototype African strain, Usutu SAAR-1776 virus, is comparatively attenuated. To understand viral pathogenesis and combat infection effectively, we sought to determine the structure of USUV SAAR-1776. Since the first structure of mature Dengue determined in 2002 using single-particle electron microscopy, higher resolution studies of flaviviruses were till recently limited to the structure of the Zika virus at 3.1 Å resolution. Here, we describe the structures of mature USUV, solved using single-particle cryo-electron microscopy to a resolution of 2.4 Å, the highest resolution for a mature flavivirus particle to-date, thus providing unprecedented detail of the structural proteins forming the icosahedral particle. We compare the USUV structures with previously determined structures of mature flaviviruses and highlight 1) the commonalities of a basic mature flavivirus particle, 2) features observed as a direct result of the high resolution of USUV structures and that may have implications for the wider field of flavivirus biology, and 3) unique features of the USUV particle that provides new knowledge for USUV pathogenesis. 

View Abstract 718

Author

Baldeep Khare, Purdue University West lafayette, IN 

Additional Author(s)

Thomas Klose, Purdue University
Qianglin Fang, Purdue University
Michael Rossmann, Purdue University IN 
Richard Kuhn, Purdue University IN 

Coffee Break


Constraining Evolution ⇒ Avoiding Drug Resistance: Lessons from Viruses

Drug resistance negatively impacts the lives of millions of patients and costs our society billions of dollars by limiting the longevity of many of our most potent drugs. Drug resistance can be caused by a change in the balance of molecular recognition events that selectively weakens inhibitor binding but maintains the biological function of the target. To reduce the likelihood of drug resistance, a detailed understanding of the target's function is necessary. Both structure at atomic resolution and evolutionarily constraints on its variation is required. This rationale was derived from our lab's experience with substrate recognition and drug resistance in HIV, HCV and most recently applying to emerging targets of HTLV-1 and SARS-CoV-2. This resulted in our development of the strategy of the substrate envelope as an explicit method to avoid drug resistance and develop robust inhibitors. We have acquired a rich and versatile experimental dataset of viral proteases, integrating alterations in both the protein sequence and the inhibitor with changes in potency and map this data to our crystallographic structures and parallel molecular dynamics in an internally consistent manner with machine learning to elucidate molecular mechanisms of drug resistance. These principals are generally applicable to other quickly evolving diseases where drug resistance is quickly evolving. 

View Abstract 755

Author

Celia Schiffer, University of Massachusetts Medical School Worcester, MA 

Deploying high-throughput protein crystallography-based drug discovery platforms to establish a structure-based drug discovery system for SARS-CoV-2 proteins

The novel SARS coronavirus (SARS-CoV-2) global pandemic has taken a strong foothold. There is a strong need for effective treatments that can be administered to infected individuals. We are helping to adopt a structure-based drug discovery approach for the discovery and development of novel inhibitors of the SARS-CoV-2 viral spike protein S1 subunit (attachment inhibitors), and inhibitors of the viral spike protein S2 subunit (fusion inhibitors). Our goal is to target conserved amino acid residues within these proteins to discover and advance molecules that may inhibit them. These molecules could serve as pan-coronavirus inhibitors and broad-spectrum antiviral therapeutics against multiple coronavirus strains and against the homologous proteins in SARS and MERS, thereby effectively creating treatments for both current and future coronavirus outbreaks. Coronaviruses (CoVs) infect humans and animals and cause a variety of diseases, primarily respiratory. There are estimated to be hundreds to thousands of CoVs in bats and other species and there have been at least three documented transmissions from animals to humans in the past 20 years leading to human viral pandemics and epidemics like COVID-19, SARS and MERS. It is only a matter of time before the next CoV hits us.
Over the past 20 years, primarily driven by high-throughput structural genomics and structural biology efforts in the USA and around the world, we have been involved in the development of instrumentation and automation for high-throughput protein X-ray crystallography, covering all stages of the gene-to-structure process. Outcomes include methods, processes, protocols, and instrumentation for high-throughput protein expression, purification, crystallization, structure determination, refinement, and analysis. Benefits have now percolated into industry and academia, influencing both structural biology and structure-based drug discovery research and have led to significant gains in efficiency and productivity at reduced cost. In addition, we have now built and deployed our novel high-throughput crystallography-based drug discovery platforms, including screening molecular compound libraries, such as fragment and scaffold libraries directly by X-ray crystallography for hit generation, which provides a direct experimental route to high quality, high reliability and high value results. Our goal is establishing a structure-based drug discovery system for the discovery and development of novel SARS-COV-2 therapeutics.
Towards this effort, we have completed determination of the highest resolution experimental X-ray crystal structures of the apo proteins of SARS-CoV-2 (a) S1 RBD (human ACE2 Receptor Binding Domain) at 1.8Å resolution; and (b) the S2 post-fusion core at 2.2Å resolution. In addition, we have also set up a biophysical assay for the S1 RBD using surface plasmon resonance. Our work resulting in high resolution experimental apo protein structures sets the stage for a target-focused structure-and biophysics-guided system for testing and validating compounds against SARS-CoV-2 S1 RBD and S2. 

View Abstract 487

Author

Debanu Das, Accelero Biostructures San Carlos, CA 

Direct visualization of SARS-CoV-2 main protease electrostatics using neutron crystallography

The SARS-CoV-2 virus that causes COVID-19 induced a worldwide economic and public health calamity in 2020. SARS-CoV-2 possesses an essential cysteine protease (Mpro) which serves as the 'heart' of viral replication and thus is a major target for small-molecule inhibitors. Structural biology strategies historically achieved atomic scale understanding of enzymes from cryogenically preserved samples using X-ray diffraction. Conventional protein X-ray crystallography studies are nevertheless hindered by the possibility of cryo-artifacts and the inability to determine protonation states, whereas neutrons provide an ideal probe to directly visualize protonation states of ionizable residues at near-physiological temperatures. A series of rapid room-temperature X-ray and neutron diffraction studies of Mpro are presented here encompassing our response to the pandemic, providing essential details about Mpro structure, function and, inhibition by reversible covalent and non-covalent inhibitors.
Pre-requisite room-temperature X-ray structures of Mpro were solved while growing the large-volume protein crystals amenable to neutron diffraction experiments. Rapid insights in the early months of the pandemic were provided by describing the inherent structural plasticity of the active site cavity [1]. We discovered that the catalytic Cys145 can be trapped in the rare peroxysulfenic acid oxidation state at physiological pH while surface cysteines remain reduced indicative of the cysteine's high reactivity [2]. Structural comparisons between clinical HCV protease inhibitors indicated how significant malleability of active site residues operate during induced fit with different inhibitor moieties [3].
The neutron crystal structure of ligand-free Mpro was determined, providing direct observation of protonation states in any cysteine protease for the first time and painting the picture of the active site electrostatics. The non-canonical catalytic dyad of Cys145-His41 exists in the reactive zwitterionic state at rest, with charged thiolate and doubly protonated imidazole side chains [4]. We determined a follow-up neutron crystal structure of deuterated Mpro in complex with a covalent inhibitor to witness the net positive charge of the active site is maintained through rearrangements of protonation states and remodeling of the active site electrostatics [5]. Neutron crystallography of Mpro showcases the importance of accurate experimental models for mechanistic, in silico, and drug design research to better understand pathogens at the atomic level. All structures and results were immediately shared with the scientific community allowing for real-time contributions to fight COVID-19.

[1] Kneller, D. W.; Phillips, G.; O'Neill, H. M.; Jedrzejczak, R.; Stols, L.; Langan, P.; Joachimiak, A.; Coates, L.; Kovalevsky, A. Structural Plasticity of SARS-CoV-2 3CL Mpro Active Site Cavity Revealed by Room Temperature X-Ray Crystallography. Nat. Commun. 2020, 11 (1), 7–12. https://doi.org/10.1038/s41467-020-16954-7.
[2] Kneller, D. W.; Phillips, G.; O'Neill, H. M.; Tan, K.; Joachimiak, A.; Coates, L.; Kovalevsky, A. Room-Temperature X-Ray Crystallography Reveals the Oxidation and Reactivity of Cysteine Residues in SARS-CoV-2 3CL M pro : Insights into Enzyme Mechanism and Drug Design . IUCrJ 2020, 7 (6). https://doi.org/10.1107/s2052252520012634.
[3] Kneller, D. W.; Galanie, S.; Phillips, G.; O'Neill, H. M.; Coates, L.; Kovalevsky, A. Malleability of the SARS-CoV-2 3CL Mpro Active Site Cavity Facilitates Binding of Clinical Antivirals. Structure 2020. https://doi.org/10.1016/j.str.2020.10.007.
[4] Kneller, D. W.; Phillips, G.; Weiss, K. L.; Pant, S.; Zhang, Q.; O'Neill, H. M.; Coates, L.; Kovalevsky, A. Unusual Zwitterionic Catalytic Site of SARS-CoV-2 Main Protease Revealed by Neutron Crystallography. J. Biol. Chem. 2020, jbc.AC120.016154. https://doi.org/10.1074/JBC.AC120.016154.
[5] Kneller, D. W.; Phillips, G.; Weiss, K. L.; Zhang, Q.; Coates, L.; Kovalevsky, A. Direct Observation of Protonation State Modulation in SARS-CoV-2 Main Protease upon Inhibitor Binding with Neutron Crystallography. J. Med. Chem. 2021. https://doi.org/10.1021/acs.jmedchem.1c00058. 

View Abstract 481

Author

Daniel Kneller, New England Biolabs Peabody, MA 

Additional Author(s)

Stephanie Galanie, Oak Ridge National Lab Oak Ridge, TN 
Gwyndalyn Phillips, Oak Ridge National Lab Oak Ridge, TN 
Leighton Coates, Oak Ridge National Laboratory Oak Ridge, TN 
Andrey Kovalevsky, Oak Ridge National Lab Oak Ridge, TN 

SARS-CoV-2 ferritin nanoparticle vaccines elicit broad SARS coronavirus immunogenicity

The need for next-generation SARS-CoV-2 vaccines has been highlighted by the rapid emergence of variants of concern (VoC), while the long-term threat of other coronaviruses further highlights the need for pan-coronavirus vaccines that can provide broad protection. Using structure-based vaccine design, we designed and characterized four categories of engineered nanoparticle CoV immunogens that recapitulate the structural and antigenic properties of prefusion Spike (S), S1 and RBD. These encompass the major antigenic regions of the S ectodomain. These immunogens were assessed in multiple animal models including mice, hamsters, and non-human primates and in all cases induced robust S-binding, ACE2-inhibition, and authentic and pseudovirus neutralizing antibodies against SARS-CoV-2.

A Spike-ferritin nanoparticle (SpFN) vaccine elicited neutralizing titers more than 20-fold higher than convalescent donor serum, following a single immunization, while RBD-Ferritin nanoparticle (RFN) immunogens elicited similar responses after two immunizations in mice. Passive transfer of IgG purified from SpFN- or RFN-immunized mice protected K18-hACE2 transgenic mice from a lethal SARS-CoV-2 virus challenge. Immunization of non-human primates, and hamsters, produced high neutralizing antibody titers, and provided robust protection against SARS-CoV-2 and VoC viral challenge in these animal models. Furthermore, SpFN- and RFN-immunization elicited ACE2 blocking activity and neutralizing ID50 antibody titers >2,000 against SARS-CoV-1, in mice, non-human primates, and hamsters, along with high magnitude neutralizing titers against the major SARS-CoV-2 VoC.

Structure-based design parameters from these SARS-CoV-2 immunogens have been translated to other coronavirus nanoparticle immunogens that are currently being tested in animal studies. Overall, these results provide a design blueprint for pan-coronavirus vaccine development. 

View Abstract 681

Author

michael joyce, Henry M. Jackson Foundation / Walter Reed Army Institute of Research washington, DC