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Host cofactors and pharmacologic ligands share an essential interface in HIV-1 capsid that is lost upon disassembly.

Price AJ, Jacques DA, McEwan WA, Fletcher AJ, Essig S, Chin JW, Halambage UD, Aiken C, James LC - PLoS Pathog. (2014)

Bottom Line: The second interface is conformationally dynamic, but binding of NUP153 or CPSF6 peptides is accommodated by only one conformation.NUP153 and CPSF6 have overlapping binding sites, but each makes unique CA interactions that, when mutated selectively, perturb cofactor dependency.These results reveal that multiple ligands share an overlapping interface in HIV-1 capsid that is lost upon viral disassembly.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory of Molecular Biology, Division of Protein and Nucleic Acid Chemistry, Cambridge, United Kingdom.

ABSTRACT
The HIV-1 capsid is involved in all infectious steps from reverse transcription to integration site selection, and is the target of multiple host cell and pharmacologic ligands. However, structural studies have been limited to capsid monomers (CA), and the mechanistic basis for how these ligands influence infection is not well understood. Here we show that a multi-subunit interface formed exclusively within CA hexamers mediates binding to linear epitopes within cellular cofactors NUP153 and CPSF6, and is competed for by the antiretroviral compounds PF74 and BI-2. Each ligand is anchored via a shared phenylalanine-glycine (FG) motif to a pocket within the N-terminal domain of one monomer, and all but BI-2 also make essential interactions across the N-terminal domain: C-terminal domain (NTD:CTD) interface to a second monomer. Dissociation of hexamer into CA monomers prevents high affinity interaction with CPSF6 and PF74, and abolishes binding to NUP153. The second interface is conformationally dynamic, but binding of NUP153 or CPSF6 peptides is accommodated by only one conformation. NUP153 and CPSF6 have overlapping binding sites, but each makes unique CA interactions that, when mutated selectively, perturb cofactor dependency. These results reveal that multiple ligands share an overlapping interface in HIV-1 capsid that is lost upon viral disassembly.

No MeSH data available.


Related in: MedlinePlus

Crystal structures of CPSF6313–327 (A) and NUP1531407–1423 (B) FG-containing peptides in complex with HIV-1 hexamer.The P6 crystal form is shown, with the six subunits of the HIV-1 capsid generated by crystallographic symmetry. The capsid is shown in a ribbon representation with each monomer colored differently. Atoms comprising the cofactor peptides are shown as spheres, with each peptide colored separately. There are six cofactor binding sites per hexamer and six bound cofactor peptides. For each structure two views are shown, related by 90°.
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ppat-1004459-g002: Crystal structures of CPSF6313–327 (A) and NUP1531407–1423 (B) FG-containing peptides in complex with HIV-1 hexamer.The P6 crystal form is shown, with the six subunits of the HIV-1 capsid generated by crystallographic symmetry. The capsid is shown in a ribbon representation with each monomer colored differently. Atoms comprising the cofactor peptides are shown as spheres, with each peptide colored separately. There are six cofactor binding sites per hexamer and six bound cofactor peptides. For each structure two views are shown, related by 90°.

Mentions: To understand how CPSF6 and NUP153 interact with hexameric CA we determined X-ray crystal structures with the same peptide ligands used in the ITC studies. Crystals of each complex were obtained in two different spacegroups, hexagonal (P6) and orthorhombic (P212121) (Table 1), similar to those reported for the uncomplexed CA hexamer [31]. The structures reveal that the binding pocket identified for CPSF6 in CA NTD is actually part of a much larger protein-protein interface that also accommodates NUP153 and is present only in assembled CA (Figure 2 and 3). The complete binding site is formed by NTD helices 3 and 4 of one CA monomer (referred to as the ‘first site’) and NTD helices 2 and 7 and CTD helices 8 and 9 from an adjacent monomer (referred to as the ‘second site’). The cofactor interface incorporates a structurally dynamic region involving CTD helices 8 and 9 that was observed to adopt two distinct conformations in the orthorhombic crystal structure of the uncomplexed hexamer (Figure 3). Comparison to CPSF6 and NUP153 complexes solved in the same orthorhombic form reveals that these cofactors selectively bind to one of these conformations, which we have termed ‘open’ (Figure 3 and Supplementary Figure S1). In the ‘open’ conformation, helices 8 and 9 are of equal length and separated by a single residue. In the alternative ‘closed’ conformation ∼1/3 of helix 9 has unwound, creating a loop of six residues (177–182) that folds back into the binding site, partially occluding it. Binding of CPSF6 and NUP153 to hexamer in the ‘closed’ conformation is prevented by steric clashes with loop177–182. A consequence of cofactor binding may therefore be to alter capsid conformational dynamics and drive equilibrium towards an ‘open’ conformation at the CTD-CTD or hexamer-hexamer interface.


Host cofactors and pharmacologic ligands share an essential interface in HIV-1 capsid that is lost upon disassembly.

Price AJ, Jacques DA, McEwan WA, Fletcher AJ, Essig S, Chin JW, Halambage UD, Aiken C, James LC - PLoS Pathog. (2014)

Crystal structures of CPSF6313–327 (A) and NUP1531407–1423 (B) FG-containing peptides in complex with HIV-1 hexamer.The P6 crystal form is shown, with the six subunits of the HIV-1 capsid generated by crystallographic symmetry. The capsid is shown in a ribbon representation with each monomer colored differently. Atoms comprising the cofactor peptides are shown as spheres, with each peptide colored separately. There are six cofactor binding sites per hexamer and six bound cofactor peptides. For each structure two views are shown, related by 90°.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214760&req=5

ppat-1004459-g002: Crystal structures of CPSF6313–327 (A) and NUP1531407–1423 (B) FG-containing peptides in complex with HIV-1 hexamer.The P6 crystal form is shown, with the six subunits of the HIV-1 capsid generated by crystallographic symmetry. The capsid is shown in a ribbon representation with each monomer colored differently. Atoms comprising the cofactor peptides are shown as spheres, with each peptide colored separately. There are six cofactor binding sites per hexamer and six bound cofactor peptides. For each structure two views are shown, related by 90°.
Mentions: To understand how CPSF6 and NUP153 interact with hexameric CA we determined X-ray crystal structures with the same peptide ligands used in the ITC studies. Crystals of each complex were obtained in two different spacegroups, hexagonal (P6) and orthorhombic (P212121) (Table 1), similar to those reported for the uncomplexed CA hexamer [31]. The structures reveal that the binding pocket identified for CPSF6 in CA NTD is actually part of a much larger protein-protein interface that also accommodates NUP153 and is present only in assembled CA (Figure 2 and 3). The complete binding site is formed by NTD helices 3 and 4 of one CA monomer (referred to as the ‘first site’) and NTD helices 2 and 7 and CTD helices 8 and 9 from an adjacent monomer (referred to as the ‘second site’). The cofactor interface incorporates a structurally dynamic region involving CTD helices 8 and 9 that was observed to adopt two distinct conformations in the orthorhombic crystal structure of the uncomplexed hexamer (Figure 3). Comparison to CPSF6 and NUP153 complexes solved in the same orthorhombic form reveals that these cofactors selectively bind to one of these conformations, which we have termed ‘open’ (Figure 3 and Supplementary Figure S1). In the ‘open’ conformation, helices 8 and 9 are of equal length and separated by a single residue. In the alternative ‘closed’ conformation ∼1/3 of helix 9 has unwound, creating a loop of six residues (177–182) that folds back into the binding site, partially occluding it. Binding of CPSF6 and NUP153 to hexamer in the ‘closed’ conformation is prevented by steric clashes with loop177–182. A consequence of cofactor binding may therefore be to alter capsid conformational dynamics and drive equilibrium towards an ‘open’ conformation at the CTD-CTD or hexamer-hexamer interface.

Bottom Line: The second interface is conformationally dynamic, but binding of NUP153 or CPSF6 peptides is accommodated by only one conformation.NUP153 and CPSF6 have overlapping binding sites, but each makes unique CA interactions that, when mutated selectively, perturb cofactor dependency.These results reveal that multiple ligands share an overlapping interface in HIV-1 capsid that is lost upon viral disassembly.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory of Molecular Biology, Division of Protein and Nucleic Acid Chemistry, Cambridge, United Kingdom.

ABSTRACT
The HIV-1 capsid is involved in all infectious steps from reverse transcription to integration site selection, and is the target of multiple host cell and pharmacologic ligands. However, structural studies have been limited to capsid monomers (CA), and the mechanistic basis for how these ligands influence infection is not well understood. Here we show that a multi-subunit interface formed exclusively within CA hexamers mediates binding to linear epitopes within cellular cofactors NUP153 and CPSF6, and is competed for by the antiretroviral compounds PF74 and BI-2. Each ligand is anchored via a shared phenylalanine-glycine (FG) motif to a pocket within the N-terminal domain of one monomer, and all but BI-2 also make essential interactions across the N-terminal domain: C-terminal domain (NTD:CTD) interface to a second monomer. Dissociation of hexamer into CA monomers prevents high affinity interaction with CPSF6 and PF74, and abolishes binding to NUP153. The second interface is conformationally dynamic, but binding of NUP153 or CPSF6 peptides is accommodated by only one conformation. NUP153 and CPSF6 have overlapping binding sites, but each makes unique CA interactions that, when mutated selectively, perturb cofactor dependency. These results reveal that multiple ligands share an overlapping interface in HIV-1 capsid that is lost upon viral disassembly.

No MeSH data available.


Related in: MedlinePlus