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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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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I03-Macromolecular Crystallography
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Goedele
Maertens
,
N. J.
Cook
,
Weifeng
Wang
,
Stephen
Hare
,
Saumya
Gupta
,
Ilker
Oztop
,
Kyeong Eun
Lee
,
Valerie
Pye
,
Ophelie
Cosnefroy
,
Ambrosius P.
Snijders
,
Vineet N.
Kewalramani
,
Ariberto
Fassati
,
Alan
Engelman
,
Peter
Cherepanov
Diamond Proposal Number(s):
[9424]
Abstract: Transportin 3 (Tnpo3, Transportin-SR2) is implicated in nuclear import of splicing factors and HIV-1 replication. Herein, we show that the majority of cellular Tnpo3 binding partners contain arginine-serine (RS) repeat domains and present crystal structures of human Tnpo3 in its free as well as GTPase Ran- and alternative splicing factor/splicing factor 2 (ASF/SF2)-bound forms. The flexible β-karyopherin fold of Tnpo3 embraces the RNA recognition motif and RS domains of the cargo. A constellation of charged residues on and around the arginine-rich helix of Tnpo3 HEAT repeat 15 engage the phosphorylated RS domain and are critical for the recognition and nuclear import of ASF/SF2. Mutations in the same region of Tnpo3 impair its interaction with the cleavage and polyadenylation specificity factor 6 (CPSF6) and its ability to support HIV-1 replication. Steric incompatibility of the RS domain and RanGTP engagement by Tnpo3 provides the mechanism for cargo release in the nucleus. Our results elucidate the structural bases for nuclear import of splicing factors and the Tnpo3–CPSF6 nexus in HIV-1 biology.
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Feb 2014
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I02-Macromolecular Crystallography
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Abstract: At the end of cell division, cytokinesis splits the cytoplasm of nascent daughter cells and partitions segregated sister genomes1, 2. To coordinate cell division with chromosome segregation, the mitotic spindle controls cytokinetic events at the cell envelope. The spindle midzone stimulates the actomyosin-driven contraction of the cleavage furrow, which proceeds until the formation of a microtubule-rich intercellular bridge with the midbody at its centre. The midbody directs the final membrane abscission reaction1, 2 and has been proposed to attach the cleavage furrow to the intercellular bridge3. How the mitotic spindle is connected to the plasma membrane during cytokinesis is not understood. Here we identify a plasma membrane tethering activity in the centralspindlin protein complex, a conserved component of the spindle midzone and midbody4. We demonstrate that the C1?domain of the centralspindlin subunit MgcRacGAP associates with the plasma membrane by interacting with polyanionic phosphoinositide lipids. Using X-ray crystallography we determine the structure of this atypical C1?domain. Mutations in the hydrophobic cap and in basic residues of the C1?domain of MgcRacGAP prevent association of the protein with the plasma membrane, and abrogate cytokinesis in human and chicken cells. Artificial membrane tethering of centralspindlin restores cell division in the absence of the C1?domain of MgcRacGAP. Although C1?domain function is dispensable for the formation of the midzone and midbody, it promotes contractility and is required for the attachment of the plasma membrane to the midbody, a long-postulated function of this organelle3. Our analysis suggests that centralspindlin links the mitotic spindle to the plasma membrane to secure the final cut during cytokinesis in animal cells.
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Dec 2012
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Mathieu
Métifiot
,
Kasthuraiah
Maddali
,
Barry C.
Johnson
,
Stephen
Hare
,
Steven J.
Smith
,
Xue Zhi
Zhao
,
Christophe
Marchand
,
Terrence R.
Burke
,
Stephen H.
Hughes
,
Peter
Cherepanov
,
Yves
Pommier
Abstract: On the basis of a series of lactam and phthalimide derivatives that inhibit HIV-1 integrase, we developed a new molecule, XZ-259, with biochemical and antiviral activities comparable to raltegravir. We determined the crystal structures of XZ-259 and four other derivatives in complex with the prototype foamy virus intasome. The compounds bind at the integrase-Mg2+-DNA interface of the integrase active site. In biochemical and antiviral assays, XZ-259 inhibits raltegravir-resistant HIV-1 integrases harboring the Y143R mutation. Molecular modeling is also presented suggesting that XZ-259 can bind in the HIV-1 intasome with its dimethyl sulfonamide group adopting two opposite orientations. Molecular dynamics analyses of the HIV-1 intasome highlight the importance of the viral DNA in drug potency
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Dec 2012
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