I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13775]
Open Access
Abstract: Integrase strand transfer inhibitors (INSTIs) block the integration step of the retroviral lifecycle and are first-line drugs used for the treatment of HIV-1/AIDS. INSTIs have a polycyclic core with heteroatom triads, chelate the metal ions at the active site, and have a halobenzyl group that interacts with viral DNA attached to the core by a flexible linker. The most broadly effective INSTIs inhibit both wild-type (WT) integrase (IN) and a variety of well-known mutants. However, because there are mutations that reduce the potency of all of the available INSTIs, new and better compounds are needed. Models based on recent structures of HIV-1 and red-capped mangabey SIV INs suggest modifications in the INSTI structures that could enhance interactions with the 3′-terminal adenosine of the viral DNA, which could improve performance against INSTI resistant mutants. We designed and tested a series of INSTIs having modifications to their naphthyridine scaffold. One of the new compounds retained good potency against an expanded panel of HIV-1 IN mutants that we tested. Our results suggest the possibility of designing inhibitors that combine the best features of the existing compounds, which could provide additional efficacy against known HIV-1 IN mutants.
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Mar 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[17221, 12579]
Open Access
Abstract: Human T-cell lymphotropic virus type 1 (HTLV-1) is a deltaretrovirus and the most oncogenic pathogen. Many of the ~20 million HTLV-1 infected people will develop severe leukaemia or an ALS-like motor disease, unless a therapy becomes available. A key step in the establishment of infection is the integration of viral genetic material into the host genome, catalysed by the retroviral integrase (IN) enzyme. Here, we use X-ray crystallography and single-particle cryo-electron microscopy to determine the structure of the functional deltaretroviral IN assembled on viral DNA ends and bound to the B56γ subunit of its human host factor, protein phosphatase 2 A. The structure reveals a tetrameric IN assembly bound to two molecules of the phosphatase via a conserved short linear motif. Insight into the deltaretroviral intasome and its interaction with the host will be crucial for understanding the pattern of integration events in infected individuals and therefore bears important clinical implications.
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Oct 2020
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9424]
Open Access
Abstract: CDC7 is an essential Ser/Thr kinase that acts upon the replicative helicase throughout the S phase of the cell cycle and is activated by DBF4. Here, we present crystal structures of a highly active human CDC7-DBF4 construct. The structures reveal a zinc-finger domain at the end of the kinase insert 2 that pins the CDC7 activation loop to motif M of DBF4 and the C lobe of CDC7. These interactions lead to ordering of the substrate-binding platform and full opening of the kinase active site. In a co-crystal structure with a mimic of MCM2 Ser40 phosphorylation target, the invariant CDC7 residues Arg373 and Arg380 engage phospho-Ser41 at substrate P+1 position, explaining the selectivity of the S-phase kinase for Ser/Thr residues followed by a pre-phosphorylated or an acidic residue. Our results clarify the role of DBF4 in activation of CDC7 and elucidate the structural basis for recognition of its preferred substrates.
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Jun 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9424]
Open Access
Abstract: Cleavage factor I mammalian (CFIm) complex, composed of cleavage and polyadenylation specificity factor 5 (CPSF5) and serine/arginine-like protein CPSF6, regulates alternative polyadenylation (APA). Loss of CFIm function results in proximal polyadenylation site usage, shortening mRNA 3′ untranslated regions (UTRs). Although CPSF6 plays additional roles in human disease, its nuclear translocation mechanism remains unresolved. Two β-karyopherins, transportin (TNPO) 1 and TNPO3, can bind CPSF6 in vitro, and we demonstrate here that while the TNPO1 binding site is dispensable for CPSF6 nuclear import, the arginine/serine (RS)-like domain (RSLD) that mediates TNPO3 binding is critical. The crystal structure of the RSLD-TNPO3 complex revealed potential CPSF6 interaction residues, which were confirmed to mediate TNPO3 binding and CPSF6 nuclear import. Both binding and nuclear import were independent of RSLD phosphorylation, though a hyperphosphorylated mimetic mutant failed to bind TNPO3 and mislocalized to the cell cytoplasm. Although hypophosphorylated CPSF6 largely supported normal polyadenylation site usage, a significant number of mRNAs harbored unnaturally extended 3′ UTRs, similar to what is observed when other APA regulators, such as CFIIm component proteins, are depleted. Our results clarify the mechanism of CPSF6 nuclear import and highlight differential roles for RSLD phosphorylation in nuclear translocation versus regulation of APA.
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Mar 2019
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Xue Zhi
Zhao
,
Steven J
Smith
,
Daniel P.
Maskell
,
Mathieu
Métifiot
,
Valerie E.
Pye
,
Katherine
Fesen
,
Christophe
Marchand
,
Yves
Pommier
,
Peter
Cherepanov
,
Stephen H
Hughes
,
Terrence R
Burke
Diamond Proposal Number(s):
[9424, 13775]
Abstract: Integrase mutations can reduce effectiveness of the first-generation FDA-approved integrase strand transfer inhibitors (INSTIs), raltegravir (RAL) and elvitegravir (EVG). The second-generation agent, dolutegravir (DTG) has enjoyed considerable clinical success; however, resistance-causing mutations that diminish the efficacy of DTG have appeared. Our current findings support and extend the substrate envelope concept that broadly effective INSTIs can be designed by filling the envelope defined by the DNA substrates. Previously, we explored 1-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamides as an INSTI scaffold, making a limited set of derivatives, and concluded that broadly effective INSTIs can be developed using this scaffold. Herein, we report an extended investigation of 6-substituents as well the first examples of 7-substituted analogs of this scaffold. While 7-substituents are not well-tolerated, we have identified novel substituents at the 6-position that are highly effective, with the best compound (6p) retaining better efficacy against a broad panel of known INSTI resistant mutants than any analogs we have previously described.
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Jul 2017
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9424, 7299]
Open Access
Abstract: ORC, Cdc6 and Cdt1 act together to load hexameric MCM, the motor of the eukaryotic replicative helicase, into double hexamers at replication origins. Here we show that Cdt1 interacts with MCM subunits Mcm2, 4 and 6, which both destabilizes the Mcm2–5 interface and inhibits MCM ATPase activity. Using X-ray crystallography, we show that Cdt1 contains two winged-helix domains in the C-terminal half of the protein and a catalytically inactive dioxygenase-related N-terminal domain, which is important for MCM loading, but not for subsequent replication. We used these structures together with single-particle electron microscopy to generate three-dimensional models of MCM complexes. These show that Cdt1 stabilizes MCM in a left-handed spiral open at the Mcm2–5 gate. We propose that Cdt1 acts as a brace, holding MCM open for DNA entry and bound to ATP until ORC–Cdc6 triggers ATP hydrolysis by MCM, promoting both Cdt1 ejection and MCM ring closure.
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Jun 2017
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9826]
Open Access
Abstract: The interactions between a retrovirus and host cell chromatin that underlie integration and provirus expression are poorly understood. The prototype foamy virus (PFV) structural protein GAG associates with chromosomes via a chromatin-binding sequence (CBS) located within its C-terminal region. Here, we show that the PFV CBS is essential and sufficient for a direct interaction with nucleosomes and present a crystal structure of the CBS bound to a mononucleosome. The CBS interacts with the histone octamer, engaging the H2A–H2B acidic patch in a manner similar to other acidic patch-binding proteins such as herpesvirus latency-associated nuclear antigen (LANA). Substitutions of the invariant arginine anchor residue in GAG result in global redistribution of PFV and macaque simian foamy virus (SFVmac) integration sites toward centromeres, dampening the resulting proviral expression without affecting the overall efficiency of integration. Our findings underscore the importance of retroviral structural proteins for integration site selection and the avoidance of genomic junkyards.
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May 2017
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Allison
Ballandras-colas
,
Daniel P.
Maskell
,
Erik
Serrao
,
Julia
Locke
,
Paolo
Swuec
,
Stefán R.
Jónsson
,
Abhay
Kotecha
,
Nicola J.
Cook
,
Valerie E.
Pye
,
Ian A.
Taylor
,
Valgerdur
Andrésdóttir
,
Alan N.
Engelman
,
Alessandro
Costa
,
Peter
Cherepanov
Diamond Proposal Number(s):
[13775]
Abstract: Retroviral integrase (IN) functions within the intasome nucleoprotein complex to catalyze insertion of viral DNA into cellular chromatin. Using cryo–electron microscopy, we now visualize the functional maedi-visna lentivirus intasome at 4.9 angstrom resolution. The intasome comprises a homo-hexadecamer of IN with a tetramer-of-tetramers architecture featuring eight structurally distinct types of IN protomers supporting two catalytically competent subunits. The conserved intasomal core, previously observed in simpler retroviral systems, is formed between two IN tetramers, with a pair of C-terminal domains from flanking tetramers completing the synaptic interface. Our results explain how HIV-1 IN, which self-associates into higher-order multimers, can form a functional intasome, reconcile the bulk of early HIV-1 IN biochemical and structural data, and provide a lentiviral platform for design of HIV-1 IN inhibitors.
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Jan 2017
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I03-Macromolecular Crystallography
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Abstract: Retroviral integrase catalyses the integration of viral DNA into host target DNA, which is an essential step in the life cycle of all retroviruses(1). Previous structural characterization of integrase-viral DNA complexes, or intasomes, from the spumavirus prototype foamy virus revealed a functional integrase tetramer(2-)5, and it is generally believed that intasomes derived from other retroviral genera use tetrameric integrase(6-9). However, the intasomes of orthoretroviruses, which include all known pathogenic species, have not been characterized structurally. Here, using single-particle cryo-electron microscopy and X-ray crystallography, we determine an unexpected octameric integrase architecture for the intasome of the betaretrovirus mouse mammary tumour virus. The structure is composed of two core integrase dimers, which interact with the viral DNA ends and structurally mimic the integrase tetramer of prototype foamy virus, and two flanking integrase dimers that engage the core structure via their integrase carboxy-terminal domains. Contrary to the belief that tetrameric integrase components are sufficient to catalyse integration, the flanking integrase dimers were necessary for mouse mammary tumour virus integrase activity. The integrase octamer solves a conundrum for betaretroviruses as well as alpharetroviruses by providing critical carboxy-terminal domains to the intasome core that cannot be provided in cis because of evolutionarily restrictive catalytic core domain-carboxy-terminal domain linker regions. The octameric architecture of the intasome of mouse mammary tumour virus provides new insight into the structural basis of retroviral DNA integration.
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Feb 2016
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Xue Zhi
Zhao
,
Steven J.
Smith
,
Daniel
Maskell
,
Mathieu
Metifiot
,
Valerie
Pye
,
Katherine
Fesen
,
Christophe
Marchand
,
Yves
Pommier
,
Peter
Cherepanov
,
Stephen H.
Hughes
,
Terrence R.
Burke
Diamond Proposal Number(s):
[9424]
Abstract: HIV integrase (IN) strand transfer inhibitors
(INSTIs) are among the newest anti-AIDS drugs; however,
mutant forms of IN can confer resistance. We developed
noncytotoxic naphthyridine-containing INSTIs that retain low
nanomolar IC50 values against HIV-1 variants harboring all of the
major INSTI-resistant mutations. We found by analyzing crystal
structures of inhibitors bound to the IN from the prototype foamy
virus (PFV) that the most successful inhibitors show striking
mimicry of the bound viral DNA prior to 3′-processing and the
bound host DNA prior to strand transfer. Using this concept of
“bi-substrate mimicry,” we developed a new broadly effective
inhibitor that not only mimics aspects of both the bound target
and viral DNA but also more completely fills the space they would normally occupy. Maximizing shape complementarity and
recapitulating structural components encompassing both of the IN DNA substrates could serve as a guiding principle for the
development of new INSTIs.
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Feb 2016
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