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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-5,10 and 14 of Vpr1-20 by CypA. A: Superimposed expanded HN-HN regions of the 2D 1H-1H NOESY spectra of sVpr1-20 in H2O:D2O (9:1, v/v) at pH 7 prior to (red signals) and after addition of 50 μl (0.1 mg) CypA (green signals). Notice the appearance of exchange peaks originating from enhanced prolyl cis/trans interconversion rate after addition of CypA. B: Superimposed expanded HN-HN regions of the 2D 1H-1H NOESY spectra of sVpr1-20 in H2O:D2O (9:1, v/v) at pH 7 after addition of 50 μl (0.1 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 5: Catalysis of isomerisation of Pro-5,10 and 14 of Vpr1-20 by CypA. A: Superimposed expanded HN-HN regions of the 2D 1H-1H NOESY spectra of sVpr1-20 in H2O:D2O (9:1, v/v) at pH 7 prior to (red signals) and after addition of 50 μl (0.1 mg) CypA (green signals). Notice the appearance of exchange peaks originating from enhanced prolyl cis/trans interconversion rate after addition of CypA. B: Superimposed expanded HN-HN regions of the 2D 1H-1H NOESY spectra of sVpr1-20 in H2O:D2O (9:1, v/v) at pH 7 after addition of 50 μl (0.1 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: In order to reveal a potential interaction of CypA with the highly conserved Pro-5, -10 and -14 of Vpr, complete series of NMR experiments allowing full assignment of the 1H chemical shifts of sVpr1-20 were recorded, followed by addition of catalytic amounts of CypA (molar ratio sVpr1-20-CypA 224:1; molar ratio sVpr1-20 proline substrate-CypA 672:1). After addition of catalytic amounts of CypA, particularly the HN-HN region of the 2D 1H-1H NOESY NMR spectrum of sVpr1-20 showed strong cis/trans exchange peaks between related protons of the all-trans isomer and various cis isomers originating from cis Pro-5, cis Pro-10 and cis Pro-14, respectively (Fig. 5A). This was further confirmed by the observation of exchange peaks for Hα of Gly-9 and those of Hδ of Pro-5 and Pro-14 (Fig. 3A). As described above for sVpr21-40 containing Pro-35, cyclosporine A was added and a further series of NMR spectra was recorded that showed the disappearance of the prolyl cis/trans exchange crosspeaks (Fig. 3B and 5B). Although Pro-5, -10 and -14 of Vpr are highly conserved [31], any interactions involving these residues have not previously been identified.


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-5,10 and 14 of Vpr1-20 by CypA. A: Superimposed expanded HN-HN regions of the 2D 1H-1H NOESY spectra of sVpr1-20 in H2O:D2O (9:1, v/v) at pH 7 prior to (red signals) and after addition of 50 μl (0.1 mg) CypA (green signals). Notice the appearance of exchange peaks originating from enhanced prolyl cis/trans interconversion rate after addition of CypA. B: Superimposed expanded HN-HN regions of the 2D 1H-1H NOESY spectra of sVpr1-20 in H2O:D2O (9:1, v/v) at pH 7 after addition of 50 μl (0.1 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 5: Catalysis of isomerisation of Pro-5,10 and 14 of Vpr1-20 by CypA. A: Superimposed expanded HN-HN regions of the 2D 1H-1H NOESY spectra of sVpr1-20 in H2O:D2O (9:1, v/v) at pH 7 prior to (red signals) and after addition of 50 μl (0.1 mg) CypA (green signals). Notice the appearance of exchange peaks originating from enhanced prolyl cis/trans interconversion rate after addition of CypA. B: Superimposed expanded HN-HN regions of the 2D 1H-1H NOESY spectra of sVpr1-20 in H2O:D2O (9:1, v/v) at pH 7 after addition of 50 μl (0.1 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: In order to reveal a potential interaction of CypA with the highly conserved Pro-5, -10 and -14 of Vpr, complete series of NMR experiments allowing full assignment of the 1H chemical shifts of sVpr1-20 were recorded, followed by addition of catalytic amounts of CypA (molar ratio sVpr1-20-CypA 224:1; molar ratio sVpr1-20 proline substrate-CypA 672:1). After addition of catalytic amounts of CypA, particularly the HN-HN region of the 2D 1H-1H NOESY NMR spectrum of sVpr1-20 showed strong cis/trans exchange peaks between related protons of the all-trans isomer and various cis isomers originating from cis Pro-5, cis Pro-10 and cis Pro-14, respectively (Fig. 5A). This was further confirmed by the observation of exchange peaks for Hα of Gly-9 and those of Hδ of Pro-5 and Pro-14 (Fig. 3A). As described above for sVpr21-40 containing Pro-35, cyclosporine A was added and a further series of NMR spectra was recorded that showed the disappearance of the prolyl cis/trans exchange crosspeaks (Fig. 3B and 5B). Although Pro-5, -10 and -14 of Vpr are highly conserved [31], any interactions involving these residues have not previously been identified.

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