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High-resolution structure of a retroviral protease folded as a monomer.

Gilski M, Kazmierczyk M, Krzywda S, Zábranská H, Cooper S, Popović Z, Khatib F, DiMaio F, Thompson J, Baker D, Pichová I, Jaskolski M - Acta Crystallogr. D Biol. Crystallogr. (2011)

Bottom Line: The flap has an unusual curled shape and a different orientation from both the open and closed states known from dimeric retropepsins.The overall fold of the protein follows the retropepsin canon, but the C(α) deviations are large and the active-site 'DTG' loop (here NTG) deviates up to 2.7 Å from the standard conformation.This structure of a monomeric retropepsin determined at high resolution (1.6 Å) provides important extra information for the design of dimerization inhibitors that might be developed as drugs for the treatment of retroviral infections, including AIDS.

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Affiliation: Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, 60-780 Poznan, Poland.

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Stereoview of superposition of the Cα atoms of the HIV-1 PR protomer (PDB entry 3hvp, red) on monomer A of M-PMV PR in the crystal structure (green). This superposition illustrates the relation of the HIV-1 PR dimer (cartoon) to the neighbouring copies of M-PMV PR monomer B in the crystal (blue and magenta). Bottom panel, view down the twofold axis of the HIV-1 PR dimer; top panel, a perpendicular view with the twofold axis vertical.
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fig5: Stereoview of superposition of the Cα atoms of the HIV-1 PR protomer (PDB entry 3hvp, red) on monomer A of M-PMV PR in the crystal structure (green). This superposition illustrates the relation of the HIV-1 PR dimer (cartoon) to the neighbouring copies of M-PMV PR monomer B in the crystal (blue and magenta). Bottom panel, view down the twofold axis of the HIV-1 PR dimer; top panel, a perpendicular view with the twofold axis vertical.

Mentions: The crystal packing is very dense, with only 28.1% of the unit-cell volume occupied by solvent (Table 1 ▶). Despite this, the two protein molecules in the asymmetric unit do not form a tight intimate dimer (see above). However, the polypeptide chains A and B do form crystal contacts (Fig. 5 ▶) that, according to PISA (Krissinel & Henrick, 2007 ▶), ‘are not strongly indicative of complex formation in solution’. These contacts bury <800 Å2 of surface area per monomer (for reference, HIV-1 PR dimerization buries ∼1700 Å2 per monomer) and are formed in a mutual fashion by interactions of the flap loop with loop-80 (Pro86–Val90). Loop-80 is an important element of the retropepsin structure as it participates in shaping the inhibitor-binding cavity. Another discernible mode of crystal packing, involving an a-translated molecule B, buries ∼400 Å2 in contacts that are formed nearly exclusively by the flap loops. This is an intriguing observation because in dimeric retropepsins the flaps also contribute to protomer interactions (in addition to the N- and C-termini and the active-site loops), especially in complexes, when they are lowered onto the bound inhibitor. In general, the lattice contacts in the present structure tend to shield from solvent the face of the molecule that is normally buried upon dimerization. When a dimer of HIV-1 PR (PDB entry 3hvp; Wlodawer et al., 1989 ▶) is superposed on molecule A of the present structure, it is obvious that the crystal-lattice aggregates of M-PMV PR are different from the functional retropepsin dimer. In particular, the active-site loops, which in the homodimer are closely associated through a ‘fireman’s grip’ and a water-mediated (or hydroxyl-mediated) contact between the catalytic aspartates, are far apart, with the Cα⋯Cα distance between the Asn26 residues being 11.4 Å. It is evident from Fig. 5 ▶ that the monomers forming the crystallo­graphic aggregates of M-PMV PR remain associated by the flaps but are ‘pulled apart’ in the active site and N-/C-terminal areas. In other words, the protomers are in close proximity and quite well juxtaposed for productive association but still do not interdigitate their N-/C-termini in a proper dimeric association. One might speculate that the dimer does not assemble by side-by-side alignment of pre-formed monomeric proteins but is more likely to arise during the folding process that involves the formation of the dimer interface (from the N- and C-termini) at an early rather than late stage, as observed for HIV-1 PR (Ishima et al., 2001 ▶).


High-resolution structure of a retroviral protease folded as a monomer.

Gilski M, Kazmierczyk M, Krzywda S, Zábranská H, Cooper S, Popović Z, Khatib F, DiMaio F, Thompson J, Baker D, Pichová I, Jaskolski M - Acta Crystallogr. D Biol. Crystallogr. (2011)

Stereoview of superposition of the Cα atoms of the HIV-1 PR protomer (PDB entry 3hvp, red) on monomer A of M-PMV PR in the crystal structure (green). This superposition illustrates the relation of the HIV-1 PR dimer (cartoon) to the neighbouring copies of M-PMV PR monomer B in the crystal (blue and magenta). Bottom panel, view down the twofold axis of the HIV-1 PR dimer; top panel, a perpendicular view with the twofold axis vertical.
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Related In: Results  -  Collection

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fig5: Stereoview of superposition of the Cα atoms of the HIV-1 PR protomer (PDB entry 3hvp, red) on monomer A of M-PMV PR in the crystal structure (green). This superposition illustrates the relation of the HIV-1 PR dimer (cartoon) to the neighbouring copies of M-PMV PR monomer B in the crystal (blue and magenta). Bottom panel, view down the twofold axis of the HIV-1 PR dimer; top panel, a perpendicular view with the twofold axis vertical.
Mentions: The crystal packing is very dense, with only 28.1% of the unit-cell volume occupied by solvent (Table 1 ▶). Despite this, the two protein molecules in the asymmetric unit do not form a tight intimate dimer (see above). However, the polypeptide chains A and B do form crystal contacts (Fig. 5 ▶) that, according to PISA (Krissinel & Henrick, 2007 ▶), ‘are not strongly indicative of complex formation in solution’. These contacts bury <800 Å2 of surface area per monomer (for reference, HIV-1 PR dimerization buries ∼1700 Å2 per monomer) and are formed in a mutual fashion by interactions of the flap loop with loop-80 (Pro86–Val90). Loop-80 is an important element of the retropepsin structure as it participates in shaping the inhibitor-binding cavity. Another discernible mode of crystal packing, involving an a-translated molecule B, buries ∼400 Å2 in contacts that are formed nearly exclusively by the flap loops. This is an intriguing observation because in dimeric retropepsins the flaps also contribute to protomer interactions (in addition to the N- and C-termini and the active-site loops), especially in complexes, when they are lowered onto the bound inhibitor. In general, the lattice contacts in the present structure tend to shield from solvent the face of the molecule that is normally buried upon dimerization. When a dimer of HIV-1 PR (PDB entry 3hvp; Wlodawer et al., 1989 ▶) is superposed on molecule A of the present structure, it is obvious that the crystal-lattice aggregates of M-PMV PR are different from the functional retropepsin dimer. In particular, the active-site loops, which in the homodimer are closely associated through a ‘fireman’s grip’ and a water-mediated (or hydroxyl-mediated) contact between the catalytic aspartates, are far apart, with the Cα⋯Cα distance between the Asn26 residues being 11.4 Å. It is evident from Fig. 5 ▶ that the monomers forming the crystallo­graphic aggregates of M-PMV PR remain associated by the flaps but are ‘pulled apart’ in the active site and N-/C-terminal areas. In other words, the protomers are in close proximity and quite well juxtaposed for productive association but still do not interdigitate their N-/C-termini in a proper dimeric association. One might speculate that the dimer does not assemble by side-by-side alignment of pre-formed monomeric proteins but is more likely to arise during the folding process that involves the formation of the dimer interface (from the N- and C-termini) at an early rather than late stage, as observed for HIV-1 PR (Ishima et al., 2001 ▶).

Bottom Line: The flap has an unusual curled shape and a different orientation from both the open and closed states known from dimeric retropepsins.The overall fold of the protein follows the retropepsin canon, but the C(α) deviations are large and the active-site 'DTG' loop (here NTG) deviates up to 2.7 Å from the standard conformation.This structure of a monomeric retropepsin determined at high resolution (1.6 Å) provides important extra information for the design of dimerization inhibitors that might be developed as drugs for the treatment of retroviral infections, including AIDS.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, 60-780 Poznan, Poland.

Show MeSH
Related in: MedlinePlus