I04-Macromolecular Crystallography
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Nitin
Hingankar
,
Suprit
Deshpande
,
Payel
Das
,
Zaigham Abbas
Rizvi
,
Constantinos Kurt
Wibmer
,
Poppy
Mashilo
,
Mohammed Yousuf
Ansari
,
Alison
Burns
,
Shawn
Barman
,
Fangzhu
Zhao
,
Sohini
Mukherjee
,
Jonathan L.
Torres
,
Souvick
Chattopadhyay
,
Farha
Mehdi
,
Jyoti
Sutar
,
Deepak Kumar
Rathore
,
Kamal
Pargai
,
Janmejay
Singh
,
Sudipta
Sonar
,
Kamini
Jakhar
,
Jyotsna
Dandotiya
,
Sankar
Bhattacharyya
,
Shailendra
Mani
,
Sweety
Samal
,
Savita
Singh
,
Pallavi
Kshetrapal
,
Ramachandran
Thiruvengadam
,
Gaurav
Batra
,
Guruprasad
Medigeshi
,
Andrew B.
Ward
,
Shinjini
Bhatnagar
,
Amit
Awasthi
,
Devin
Sok
,
Jayanta
Bhattacharya
Diamond Proposal Number(s):
[28402]
Open Access
Abstract: Although efficacious vaccines have significantly reduced the morbidity and mortality of COVID-19, there remains an unmet medical need for treatment options, which monoclonal antibodies (mAbs) can potentially fill. This unmet need is exacerbated by the emergence and spread of SARS-CoV-2 variants of concern (VOCs) that have shown some resistance to vaccine responses. Here we report the isolation of five neutralizing mAbs from an Indian convalescent donor, out of which two (THSC20.HVTR04 and THSC20.HVTR26) showed potent neutralization of SARS-CoV-2 VOCs at picomolar concentrations, including the Delta variant (B.1.617.2). One of these (THSC20.HVTR26) also retained activity against the Omicron variant. These two mAbs target non-overlapping epitopes on the receptor-binding domain (RBD) of the spike protein and prevent virus attachment to its host receptor, human angiotensin converting enzyme-2 (hACE2). Furthermore, the mAb cocktail demonstrated protection against the Delta variant at low antibody doses when passively administered in the K18 hACE2 transgenic mice model, highlighting their potential as a cocktail for prophylactic and therapeutic applications. Developing the capacity to rapidly discover and develop mAbs effective against highly transmissible pathogens like coronaviruses at a local level, especially in a low- and middle-income country (LMIC) such as India, will enable prompt responses to future pandemics as an important component of global pandemic preparedness.
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Apr 2022
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[15916, 21426]
Open Access
Abstract: The HIV capsid self-assembles a protective conical shell that simultaneously prevents host sensing whilst permitting the import of nucleotides to drive DNA synthesis. This is accomplished through the construction of dynamic, highly charged pores at the centre of each capsid multimer. The clustering of charges required for dNTP import is strongly destabilising and it is proposed that HIV uses the metabolite IP6 to coordinate the pore during assembly. Here we have investigated the role of inositol phosphates in coordinating a ring of positively charged lysine residues (K25) that forms at the base of the capsid pore. We show that whilst IP5, which can functionally replace IP6, engages an arginine ring (R18) at the top of the pore, the lysine ring simultaneously binds a second IP5 molecule. Dose dependent removal of K25 from the pore severely inhibits HIV infection and concomitantly prevents DNA synthesis. Cryo-tomography reveals that K25A virions have a severe assembly defect that inhibits the formation of mature capsid cones. Monitoring both the kinetics and morphology of capsids assembled in vitro reveals that while mutation K25A can still form tubes, the ability of IP6 to drive assembly of capsid cones has been lost. Finally, in single molecule TIRF microscopy experiments, capsid lattices in permeabilised K25 mutant virions are rapidly lost and cannot be stabilised by IP6. These results suggest that the coordination of IP6 by a second charged ring in mature hexamers drives the assembly of conical capsids capable of reverse transcription and infection.
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Feb 2021
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I03-Macromolecular Crystallography
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Open Access
Abstract: The picornaviruses coxsackievirus A24 variant (CVA24v) and enterovirus 70 (EV70) cause continued outbreaks and pandemics of acute hemorrhagic conjunctivitis (AHC), a highly contagious eye disease against which neither vaccines nor antiviral drugs are currently available. Moreover, these viruses can cause symptoms in the cornea, upper respiratory tract, and neurological impairments such as acute flaccid paralysis. EV70 and CVA24v are both known to use 5-N-acetylneuraminic acid (Neu5Ac) for cell attachment, thus providing a putative link between the glycan receptor specificity and cell tropism and disease. We report the structures of an intact human picornavirus in complex with a range of glycans terminating in Neu5Ac. We determined the structure of the CVA24v to 1.40 Å resolution, screened different glycans bearing Neu5Ac for CVA24v binding, and structurally characterized interactions with candidate glycan receptors. Biochemical studies verified the relevance of the binding site and demonstrated a preference of CVA24v for α2,6-linked glycans. This preference can be rationalized by molecular dynamics simulations that show that α2,6-linked glycans can establish more contacts with the viral capsid. Our results form an excellent platform for the design of antiviral compounds to prevent AHC.
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Oct 2014
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18565, 13467]
Open Access
Abstract: Accelerated gene evolution is a hallmark of pathogen adaptation and specialization following host-jumps. However, the molecular processes associated with adaptive evolution between host-specific lineages of a multihost plant pathogen remain poorly understood. In the blast fungus Magnaporthe oryzae (Syn. Pyricularia oryzae), host specialization on different grass hosts is generally associated with dynamic patterns of gain and loss of virulence effector genes that tend to define the distinct genetic lineages of this pathogen. Here, we unravelled the biochemical and structural basis of adaptive evolution of APikL2, an exceptionally conserved paralog of the well-studied rice-lineage specific effector AVR-Pik. Whereas AVR-Pik and other members of the six-gene AVR-Pik family show specific patterns of presence/absence polymorphisms between grass-specific lineages of M. oryzae, APikL2 stands out by being ubiquitously present in all blast fungus lineages from 13 different host species. Using biochemical, biophysical and structural biology methods, we show that a single aspartate to asparagine polymorphism expands the binding spectrum of APikL2 to host proteins of the heavy-metal associated (HMA) domain family. This mutation maps to one of the APikL2-HMA binding interfaces and contributes to an altered hydrogen-bonding network. By combining phylogenetic ancestral reconstruction with an analysis of the structural consequences of allelic diversification, we revealed a common mechanism of effector specialization in the AVR-Pik/APikL2 family that involves two major HMA-binding interfaces. Together, our findings provide a detailed molecular evolution and structural biology framework for diversification and adaptation of a fungal pathogen effector family following host-jumps.
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Nov 2021
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I03-Macromolecular Crystallography
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David C.
Goldstone
,
Thomas G.
Flower
,
Neil J.
Ball
,
Marta
Sanz-Ramos
,
Melvyn W.
Yap
,
Roksana
Ogrodowicz
,
Nicole
Stanke
,
Juliane
Reh
,
Dirk
Lindemann
,
Jonathan P.
Stoye
,
Ian A.
Taylor
Open Access
Abstract: The Spumaretrovirinae, or foamyviruses (FVs) are complex retroviruses that infect many species of monkey and ape. Although FV infection is apparently benign, trans-species zoonosis is commonplace and has resulted in the isolation of the Prototypic Foamy Virus (PFV) from human sources and the potential for germ-line transmission.
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May 2013
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14739]
Open Access
Abstract: The dUTPase (Dut) enzymes, encoded by almost all free-living organisms and some viruses, prevent the misincorporation of uracil into DNA. We previously proposed that trimeric Duts are regulatory proteins involved in different cellular processes; including the phage-mediated transfer of the Staphylococcus aureus pathogenicity island SaPIbov1. Recently, it has been shown that the structurally unrelated dimeric Dut encoded by phage ϕNM1 is similarly able to mobilize SaPIbov1, suggesting dimeric Duts could also be regulatory proteins. How this is accomplished remains unsolved. Here, using in vivo, biochemical and structural approaches, we provide insights into the signaling mechanism used by the dimeric Duts to induce the SaPIbov1 cycle. As reported for the trimeric Duts, dimeric Duts contain an extremely variable region, here named domain VI, which is involved in the regulatory capacity of these enzymes. Remarkably, our results also show that the dimeric Dut signaling mechanism is modulated by dUTP, as with the trimeric Duts. Overall, our results demonstrate that although unrelated both in sequence and structure, dimeric and trimeric Duts control SaPI transfer by analogous mechanisms, representing a fascinating example of convergent evolution. This conserved mode of action highlights the biological significance of Duts as regulatory molecules.
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Sep 2017
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Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[19832]
Open Access
Abstract: Nipah and its close relative Hendra are highly pathogenic zoonotic viruses, storing their ssRNA genome in a helical nucleocapsid assembly formed by the N protein, a major viral immunogen. Here, we report the first cryoEM structure for a Henipavirus RNA-bound nucleocapsid assembly, at 3.5 Å resolution. The helical assembly is stabilised by previously undefined N- and C-terminal segments, contributing to subunit-subunit interactions. RNA is wrapped around the nucleocapsid protein assembly with a periodicity of six nucleotides per protomer, in the “3-bases-in, 3-bases-out” conformation, with protein plasticity enabling non-sequence specific interactions. The structure reveals commonalities in RNA binding pockets and in the conformation of bound RNA, not only with members of the Paramyxoviridae family, but also with the evolutionarily distant Filoviridae Ebola virus. Significant structural differences with other Paramyxoviridae members are also observed, particularly in the position and length of the exposed α-helix, residues 123–139, which may serve as a valuable epitope for surveillance and diagnostics.
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Jul 2021
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15916]
Open Access
Abstract: Hantaviruses are important emerging human pathogens and are the causative agents of serious diseases in humans with high mortality rates. Like other members in the Bunyaviridae family their M segment encodes two glycoproteins, GN and GC, which are responsible for the early events of infection. Hantaviruses deliver their tripartite genome into the cytoplasm by fusion of the viral and endosomal membranes in response to the reduced pH of the endosome. Unlike phleboviruses (e.g. Rift valley fever virus), that have an icosahedral glycoprotein envelope, hantaviruses display a pleomorphic virion morphology as GN and GC assemble into spikes with apparent four-fold symmetry organized in a grid-like pattern on the viral membrane. Here we present the crystal structure of glycoprotein C (GC) from Puumala virus (PUUV), a representative member of the Hantavirus genus. The crystal structure shows GC as the membrane fusion effector of PUUV and it presents a class II membrane fusion protein fold. Furthermore, GC was crystallized in its post-fusion trimeric conformation that until now had been observed only in Flavi- and Togaviridae family members. The PUUV GC structure together with our functional data provides intriguing evolutionary and mechanistic insights into class II membrane fusion proteins and reveals new targets for membrane fusion inhibitors against these important pathogens
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Oct 2016
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Bruno
Correia
,
Sofia A.
Cerqueira
,
Chantal
Beauchemin
,
Marta
Pires De Miranda
,
Shijun
Li
,
Rajesh
Ponnusamy
,
Lenia
Rodrigues
,
Thomas R.
Schneider
,
Maria A.
Carrondo
,
Kenneth M.
Kaye
,
J. Pedro
Simas
,
Colin E.
Mcvey
Diamond Proposal Number(s):
[8851]
Open Access
Abstract: Latency-associated nuclear antigen (LANA) mediates γ2-herpesvirus genome persistence and regulates transcription. We describe the crystal structure of the murine gammaherpesvirus-68 LANA C-terminal domain at 2.2 Å resolution. The structure reveals an alpha-beta fold that assembles as a dimer, reminiscent of Epstein-Barr virus EBNA1. A predicted DNA binding surface is present and opposite this interface is a positive electrostatic patch. Targeted DNA recognition substitutions eliminated DNA binding, while certain charged patch mutations reduced bromodomain protein, BRD4, binding. Virus containing LANA abolished for DNA binding was incapable of viable latent infection in mice. Virus with mutations at the charged patch periphery exhibited substantial deficiency in expansion of latent infection, while central region substitutions had little effect. This deficiency was independent of BRD4. These results elucidate the LANA DNA binding domain structure and reveal a unique charged region that exerts a critical role in viral latent infection, likely acting through a host cell protein(s).
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Oct 2013
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I04-Macromolecular Crystallography
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Steven
Johnson
,
Lionel
Tan
,
Stijn
Van Der Veen
,
Joseph
Caesar
,
Elena
Goicoechea De Jorge
,
Rachel J.
Harding
,
Xilian
Bai
,
Rachel M.
Exley
,
Philip N.
Ward
,
Nicola
Ruivo
,
Kaushali
Trivedi
,
Elspeth
Cumber
,
Rhian
Jones
,
Luke
Newham
,
David
Staunton
,
Rafael
Ufret-Vincenty
,
Ray
Borrow
,
Matthew C.
Pickering
,
Susan
Lea
,
Christoph M.
Tang
Open Access
Abstract: Neisseria meningitis remains a leading cause of sepsis and meningitis, and vaccines are required to prevent infections by this important human pathogen. Factor H binding protein (fHbp) is a key antigen that elicits protective immunity against the meningococcus and recruits the host complement regulator, fH. As the high affinity interaction between fHbp and fH could impair immune responses, we sought to identify non-functional fHbps that could act as effective immunogens. This was achieved by alanine substitution of fHbps from all three variant groups (V1, V2 and V3 fHbp) of the protein; while some residues affected fH binding in each variant group, the distribution of key amino underlying the interaction with fH differed between the V1, V2 and V3 proteins. The atomic structure of V3 fHbp in complex with fH and of the C-terminal barrel of V2 fHbp provide explanations to the differences in the precise nature of their interactions with fH, and the instability of the V2 protein. To develop transgenic models to assess the efficacy of non-functional fHbps, we determined the structural basis of the low level of interaction between fHbp and murine fH; in addition to changes in amino acids in the fHbp binding site, murine fH has a distinct conformation compared with the human protein that would sterically inhibit binding to fHbp. Non-functional V1 fHbps were further characterised by binding and structural studies, and shown in non-transgenic and transgenic mice (expressing chimeric fH that binds fHbp and precisely regulates complement system) to retain their immunogenicity. Our findings provide a catalogue of non-functional fHbps from all variant groups that can be included in new generation meningococcal vaccines, and establish proof-in-principle for clinical studies to compare their efficacy with wild-type fHbps.
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Oct 2012
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