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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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Presence of cis and trans proline isomers in N-terminal sVpr. A1: Fingerprint region of the 2D 1H-1H TOCSY spectrum showing the assigned spin systems of residues close to proline residues in sVpr(1-20) and A2: 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. B1: Expanded Hα-Hβ region of the 2D 1H-1H TOCSY spectrum of sVpr21-40 in H2O:D2O (9:1, v/v) at pH 7 showing the signals of trans and cis Pro-35. B2: 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.
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Figure 2: Presence of cis and trans proline isomers in N-terminal sVpr. A1: Fingerprint region of the 2D 1H-1H TOCSY spectrum showing the assigned spin systems of residues close to proline residues in sVpr(1-20) and A2: 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. B1: Expanded Hα-Hβ region of the 2D 1H-1H TOCSY spectrum of sVpr21-40 in H2O:D2O (9:1, v/v) at pH 7 showing the signals of trans and cis Pro-35. B2: 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.

Mentions: In an initial series of 2D NMR experiments (2D 1H TOCSY, ROESY and NOESY NMR spectra), the 1H chemical shifts of the all-trans isomers of sVpr1-20, sVpr21-40 and sVpr25-40 in aqueous phosphate buffer solution at pH 7 containing 10% v/v D2O were completely assigned (Additional file 1, Table S1, Additional file 1, Table S2, Additional file 1, Table S3, Additional file 1, Table S4, Additional file 1, Table S5, Additional file 1, Table S6 and Additional file 1, Table S7). In accordance with previous investigations in aqueous organic solvent [19] the NMR spectra of sVpr1-20 revealed two sets of 1H NMR signals originating from the same residues in the sequence (Fig. 2). In each case one set of signals was more intense than the second set, and the corresponding amidic protons showed significant chemical shift differences. The fact that the residues showing the largest shift differences were those either adjacent to or very close to each of the Pro residues indicated that the weaker sets of signals in each case arose from the cis isomers of the Pro residues. The identification of sets of signals for Ala-4, Gly-9 and Tyr-15 (Fig. 2A), in addition to the 1H NMR signals originating from the cis isomers of Pro-5 and 14 (Fig. 3), is a strong indication that all Pro residues are involved in such processes under the conditions used. Similarly, 1H NMR signals originating from the cis isomer of Pro-35 were observed in the 2D 1H NMR spectra of sVpr21-40 (Fig. 2B) and sVpr25-40.


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)

Presence of cis and trans proline isomers in N-terminal sVpr. A1: Fingerprint region of the 2D 1H-1H TOCSY spectrum showing the assigned spin systems of residues close to proline residues in sVpr(1-20) and A2: 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. B1: Expanded Hα-Hβ region of the 2D 1H-1H TOCSY spectrum of sVpr21-40 in H2O:D2O (9:1, v/v) at pH 7 showing the signals of trans and cis Pro-35. B2: 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Presence of cis and trans proline isomers in N-terminal sVpr. A1: Fingerprint region of the 2D 1H-1H TOCSY spectrum showing the assigned spin systems of residues close to proline residues in sVpr(1-20) and A2: 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. B1: Expanded Hα-Hβ region of the 2D 1H-1H TOCSY spectrum of sVpr21-40 in H2O:D2O (9:1, v/v) at pH 7 showing the signals of trans and cis Pro-35. B2: 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.
Mentions: In an initial series of 2D NMR experiments (2D 1H TOCSY, ROESY and NOESY NMR spectra), the 1H chemical shifts of the all-trans isomers of sVpr1-20, sVpr21-40 and sVpr25-40 in aqueous phosphate buffer solution at pH 7 containing 10% v/v D2O were completely assigned (Additional file 1, Table S1, Additional file 1, Table S2, Additional file 1, Table S3, Additional file 1, Table S4, Additional file 1, Table S5, Additional file 1, Table S6 and Additional file 1, Table S7). In accordance with previous investigations in aqueous organic solvent [19] the NMR spectra of sVpr1-20 revealed two sets of 1H NMR signals originating from the same residues in the sequence (Fig. 2). In each case one set of signals was more intense than the second set, and the corresponding amidic protons showed significant chemical shift differences. The fact that the residues showing the largest shift differences were those either adjacent to or very close to each of the Pro residues indicated that the weaker sets of signals in each case arose from the cis isomers of the Pro residues. The identification of sets of signals for Ala-4, Gly-9 and Tyr-15 (Fig. 2A), in addition to the 1H NMR signals originating from the cis isomers of Pro-5 and 14 (Fig. 3), is a strong indication that all Pro residues are involved in such processes under the conditions used. Similarly, 1H NMR signals originating from the cis isomer of Pro-35 were observed in the 2D 1H NMR spectra of sVpr21-40 (Fig. 2B) and sVpr25-40.

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