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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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Characterization of CypA binding region of the N-terminus of Vpr. Synthetic Vpr30-40 (A), Vpr32-38 (B), Vpr33-37(C, D) were tested for binding to immobilized recombinant CypA using SPR biosensor system. Individual peptides were injected at concentration ranging from 0-400 μM over CM5 chip immobilized with 980 RU CypA. The curves were best fit to a two state model, and ka1, ka2, kd1, kd2 and KD were calculated for the respective sensograms (Table 1.).
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Figure 8: Characterization of CypA binding region of the N-terminus of Vpr. Synthetic Vpr30-40 (A), Vpr32-38 (B), Vpr33-37(C, D) were tested for binding to immobilized recombinant CypA using SPR biosensor system. Individual peptides were injected at concentration ranging from 0-400 μM over CM5 chip immobilized with 980 RU CypA. The curves were best fit to a two state model, and ka1, ka2, kd1, kd2 and KD were calculated for the respective sensograms (Table 1.).

Mentions: To identify and determine the specific binding region of N-terminal Vpr to CypA, SPR studies of the shorter Vpr peptides sVpr30-40, sVpr32-38 and sVpr33-37 were performed. The sensograms revealed that sVpr30-40and sVpr32-38 maintain the strong binding to CypA similar to that of longer N-terminal Vpr peptides (Fig. 8). In contrast, the shortest peptide sVpr33-37 binds considerably weaker. The remarkably weaker response of the binding curves of sVpr33-37 to CypA compared with the longer peptides, demonstrates that the shortest peptide sequence maintaining strong binding is the heptapeptide sVpr32-38. Thus the seven-residue motif RHFPRIW centred at Pro-35 defines the region for strong specific binding to CypA. In keeping with our findings, Zander et al. [9] and Ardon et al. [10] reported that CypA co-immunoprecipitates with wild-type Vpr, while mutation of Pro-35 caused loss of this phenomenon.


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)

Characterization of CypA binding region of the N-terminus of Vpr. Synthetic Vpr30-40 (A), Vpr32-38 (B), Vpr33-37(C, D) were tested for binding to immobilized recombinant CypA using SPR biosensor system. Individual peptides were injected at concentration ranging from 0-400 μM over CM5 chip immobilized with 980 RU CypA. The curves were best fit to a two state model, and ka1, ka2, kd1, kd2 and KD were calculated for the respective sensograms (Table 1.).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Characterization of CypA binding region of the N-terminus of Vpr. Synthetic Vpr30-40 (A), Vpr32-38 (B), Vpr33-37(C, D) were tested for binding to immobilized recombinant CypA using SPR biosensor system. Individual peptides were injected at concentration ranging from 0-400 μM over CM5 chip immobilized with 980 RU CypA. The curves were best fit to a two state model, and ka1, ka2, kd1, kd2 and KD were calculated for the respective sensograms (Table 1.).
Mentions: To identify and determine the specific binding region of N-terminal Vpr to CypA, SPR studies of the shorter Vpr peptides sVpr30-40, sVpr32-38 and sVpr33-37 were performed. The sensograms revealed that sVpr30-40and sVpr32-38 maintain the strong binding to CypA similar to that of longer N-terminal Vpr peptides (Fig. 8). In contrast, the shortest peptide sVpr33-37 binds considerably weaker. The remarkably weaker response of the binding curves of sVpr33-37 to CypA compared with the longer peptides, demonstrates that the shortest peptide sequence maintaining strong binding is the heptapeptide sVpr32-38. Thus the seven-residue motif RHFPRIW centred at Pro-35 defines the region for strong specific binding to CypA. In keeping with our findings, Zander et al. [9] and Ardon et al. [10] reported that CypA co-immunoprecipitates with wild-type Vpr, while mutation of Pro-35 caused loss of this phenomenon.

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