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The intriguing cyclophilin A-HIV-1 Vpr interaction: prolyl cis/trans isomerisation catalysis and specific binding.

Solbak SM, Reksten TR, Wray V, Bruns K, Horvli O, Raae AJ, Henklein P, Henklein P, Röder R, Mitzner D, Schubert U, Fossen T - BMC Struct. Biol. (2010)

Bottom Line: Previously suggested models depicting CypA as a chaperone that plays a role in HIV-1 virulence are now supported by our data.In detail the SPR data of this interaction were compatible with a two-state binding interaction model that involves a conformational change during binding.This is in accord with the structural changes observed by NMR suggesting CypA catalyzes the prolyl cis/trans interconversion during binding to the RHFP35RIW motif of N-terminal Vpr.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, University of Bergen, N-5007 Bergen, Norway.

ABSTRACT

Background: Cyclophilin A (CypA) represents a potential target for antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication, although the mechanism through which CypA modulates HIV-1 infectivity still remains unclear. The interaction of HIV-1 viral protein R (Vpr) with the human peptidyl prolyl isomerase CypA is known to occur in vitro and in vivo. However, the nature of the interaction of CypA with Pro-35 of N-terminal Vpr has remained undefined.

Results: Characterization of the interactions of human CypA with N-terminal peptides of HIV-1 Vpr has been achieved using a combination of nuclear magnetic resonace (NMR) exchange spectroscopy and surface plasmon resonance spectroscopy (SPR). NMR data at atomic resolution indicate prolyl cis/trans isomerisation of the highly conserved proline residues Pro-5, -10, -14 and -35 of Vpr are catalyzed by human CypA and require only very low concentrations of the isomerase relative to that of the peptide substrates. Of the N-terminal peptides of Vpr only those containing Pro-35 bind to CypA in a biosensor assay. SPR studies of specific N-terminal peptides with decreasing numbers of residues revealed that a seven-residue motif centred at Pro-35 consisting of RHFPRIW, which under membrane-like solution conditions comprises the loop region connecting helix 1 and 2 of Vpr and the two terminal residues of helix 1, is sufficient to maintain strong specific binding.

Conclusions: Only N-terminal peptides of Vpr containing Pro-35, which appears to be vital for manifold functions of Vpr, bind to CypA in a biosensor assay. This indicates that Pro-35 is essential for a specific CypA-Vpr binding interaction, in contrast to the general prolyl cis/trans isomerisation observed for all proline residues of Vpr, which only involve transient enzyme-substrate interactions. Previously suggested models depicting CypA as a chaperone that plays a role in HIV-1 virulence are now supported by our data. In detail the SPR data of this interaction were compatible with a two-state binding interaction model that involves a conformational change during binding. This is in accord with the structural changes observed by NMR suggesting CypA catalyzes the prolyl cis/trans interconversion during binding to the RHFP35RIW motif of N-terminal Vpr.

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Catalysis of isomerisation of Pro-35 of Vpr21-40 by CypA. A, C, E: Superimposed expanded Hα-Hβ regions of the 2D 1H-1H NOESY spectra of sVpr21-40 in H2O:D2O (9:1, v/v) at pH 7 prior to (red signals) and after addition of 100 μl (0.2 mg) CypA (green signals). Notice the appearance of exchange peaks originating from enhanced prolyl cis/trans interconversion rate after addition of CypA. B, D, F: Superimposed expanded Hα-Hβ region of the 2D 1H-1H NOESY spectra of sVpr21-40 in H2O:D2O (9:1, v/v) at pH 7 after addition of 100 μl (0.2 mg) CypA (green signals) and after additional addition of 5 μl (0.1 mg) cyclosporine A (red signals). Notice that the prolyl cis/trans related exchange peaks detected after addition of CypA disappear after addition of cyclosporine A.
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Figure 4: Catalysis of isomerisation of Pro-35 of Vpr21-40 by CypA. A, C, E: Superimposed expanded Hα-Hβ regions of the 2D 1H-1H NOESY spectra of sVpr21-40 in H2O:D2O (9:1, v/v) at pH 7 prior to (red signals) and after addition of 100 μl (0.2 mg) CypA (green signals). Notice the appearance of exchange peaks originating from enhanced prolyl cis/trans interconversion rate after addition of CypA. B, D, F: Superimposed expanded Hα-Hβ region of the 2D 1H-1H NOESY spectra of sVpr21-40 in H2O:D2O (9:1, v/v) at pH 7 after addition of 100 μl (0.2 mg) CypA (green signals) and after additional addition of 5 μl (0.1 mg) cyclosporine A (red signals). Notice that the prolyl cis/trans related exchange peaks detected after addition of CypA disappear after addition of cyclosporine A.

Mentions: When the complete series of NMR experiments allowing full assignment of the 1H chemical shifts of sVpr21-40 were recorded, CypA was added to the peptide solution at a molar ratio sVpr21-40-CypA 140:1, and analogous NMR experiments were then recorded. In the presence of catalytic amounts of CypA, strong exchange peaks between related signals of the cis Pro-35 and trans-Pro-35 isomer of sVpr21-40 were observed (Fig. 4A, C and 4E). In particular, strong exchange peaks between cis-Hα/trans-Hα and cis-Hδ/trans-Hδ of Pro-35 were observed in the NOESY spectra after addition of catalytic amounts of CypA.


The intriguing cyclophilin A-HIV-1 Vpr interaction: prolyl cis/trans isomerisation catalysis and specific binding.

Solbak SM, Reksten TR, Wray V, Bruns K, Horvli O, Raae AJ, Henklein P, Henklein P, Röder R, Mitzner D, Schubert U, Fossen T - BMC Struct. Biol. (2010)

Catalysis of isomerisation of Pro-35 of Vpr21-40 by CypA. A, C, E: Superimposed expanded Hα-Hβ regions of the 2D 1H-1H NOESY spectra of sVpr21-40 in H2O:D2O (9:1, v/v) at pH 7 prior to (red signals) and after addition of 100 μl (0.2 mg) CypA (green signals). Notice the appearance of exchange peaks originating from enhanced prolyl cis/trans interconversion rate after addition of CypA. B, D, F: Superimposed expanded Hα-Hβ region of the 2D 1H-1H NOESY spectra of sVpr21-40 in H2O:D2O (9:1, v/v) at pH 7 after addition of 100 μl (0.2 mg) CypA (green signals) and after additional addition of 5 μl (0.1 mg) cyclosporine A (red signals). Notice that the prolyl cis/trans related exchange peaks detected after addition of CypA disappear after addition of cyclosporine A.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2959089&req=5

Figure 4: Catalysis of isomerisation of Pro-35 of Vpr21-40 by CypA. A, C, E: Superimposed expanded Hα-Hβ regions of the 2D 1H-1H NOESY spectra of sVpr21-40 in H2O:D2O (9:1, v/v) at pH 7 prior to (red signals) and after addition of 100 μl (0.2 mg) CypA (green signals). Notice the appearance of exchange peaks originating from enhanced prolyl cis/trans interconversion rate after addition of CypA. B, D, F: Superimposed expanded Hα-Hβ region of the 2D 1H-1H NOESY spectra of sVpr21-40 in H2O:D2O (9:1, v/v) at pH 7 after addition of 100 μl (0.2 mg) CypA (green signals) and after additional addition of 5 μl (0.1 mg) cyclosporine A (red signals). Notice that the prolyl cis/trans related exchange peaks detected after addition of CypA disappear after addition of cyclosporine A.
Mentions: When the complete series of NMR experiments allowing full assignment of the 1H chemical shifts of sVpr21-40 were recorded, CypA was added to the peptide solution at a molar ratio sVpr21-40-CypA 140:1, and analogous NMR experiments were then recorded. In the presence of catalytic amounts of CypA, strong exchange peaks between related signals of the cis Pro-35 and trans-Pro-35 isomer of sVpr21-40 were observed (Fig. 4A, C and 4E). In particular, strong exchange peaks between cis-Hα/trans-Hα and cis-Hδ/trans-Hδ of Pro-35 were observed in the NOESY spectra after addition of catalytic amounts of CypA.

Bottom Line: Previously suggested models depicting CypA as a chaperone that plays a role in HIV-1 virulence are now supported by our data.In detail the SPR data of this interaction were compatible with a two-state binding interaction model that involves a conformational change during binding.This is in accord with the structural changes observed by NMR suggesting CypA catalyzes the prolyl cis/trans interconversion during binding to the RHFP35RIW motif of N-terminal Vpr.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, University of Bergen, N-5007 Bergen, Norway.

ABSTRACT

Background: Cyclophilin A (CypA) represents a potential target for antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication, although the mechanism through which CypA modulates HIV-1 infectivity still remains unclear. The interaction of HIV-1 viral protein R (Vpr) with the human peptidyl prolyl isomerase CypA is known to occur in vitro and in vivo. However, the nature of the interaction of CypA with Pro-35 of N-terminal Vpr has remained undefined.

Results: Characterization of the interactions of human CypA with N-terminal peptides of HIV-1 Vpr has been achieved using a combination of nuclear magnetic resonace (NMR) exchange spectroscopy and surface plasmon resonance spectroscopy (SPR). NMR data at atomic resolution indicate prolyl cis/trans isomerisation of the highly conserved proline residues Pro-5, -10, -14 and -35 of Vpr are catalyzed by human CypA and require only very low concentrations of the isomerase relative to that of the peptide substrates. Of the N-terminal peptides of Vpr only those containing Pro-35 bind to CypA in a biosensor assay. SPR studies of specific N-terminal peptides with decreasing numbers of residues revealed that a seven-residue motif centred at Pro-35 consisting of RHFPRIW, which under membrane-like solution conditions comprises the loop region connecting helix 1 and 2 of Vpr and the two terminal residues of helix 1, is sufficient to maintain strong specific binding.

Conclusions: Only N-terminal peptides of Vpr containing Pro-35, which appears to be vital for manifold functions of Vpr, bind to CypA in a biosensor assay. This indicates that Pro-35 is essential for a specific CypA-Vpr binding interaction, in contrast to the general prolyl cis/trans isomerisation observed for all proline residues of Vpr, which only involve transient enzyme-substrate interactions. Previously suggested models depicting CypA as a chaperone that plays a role in HIV-1 virulence are now supported by our data. In detail the SPR data of this interaction were compatible with a two-state binding interaction model that involves a conformational change during binding. This is in accord with the structural changes observed by NMR suggesting CypA catalyzes the prolyl cis/trans interconversion during binding to the RHFP35RIW motif of N-terminal Vpr.

Show MeSH
Related in: MedlinePlus