Catalysis of cis/trans isomerization in native HIV-1 capsid by human cyclophilin A

Abstract

Packaging of cyclophilin A (CypA) into HIV-1 virions is essential for efficient replication; however, the reason for this is unknown. Incorporation is mediated through binding to the Gly-89-Pro-90 peptide bond of the N-terminal domain of HIV-1 capsid (CA(N)). Despite the fact that CypA is a peptidyl-prolyl cis/trans isomerase, catalytic activity on CA(N) has not been observed previously. We show here, using NMR exchange spectroscopy, that CypA does not only bind to CA(N) but also catalyzes efficiently the cis/trans isomerization of the Gly-89-Pro-90 peptide bond. In addition, conformational changes in CA(N) distal to the CypA binding loop are observed on CypA binding and catalysis. The results provide experimental evidence for efficient CypA catalysis on a natively folded and biologically relevant protein substrate.

Figure 1

CypA catalysis of cis/trans isomerization at G89-P90 in folded CA^N. Expansion of ¹H-¹⁵N heteronuclear exchange spectra showing the amide signal of G89 for the trans and cis isomer, respectively. Chemical exchange between the cis and trans conformation of G89-P90 is slow in the absence of CypA (A) and is accelerated in the presence of catalytic amounts of CypA, as indicated by the appearance of exchange peaks between the cis and the trans auto peaks (B and C). The intensity of the exchange peak increases with longer mixing time and concurrently the less abundant cis auto peak decreases because of additional loss in magnetization from chemical exchange and longitudinal relaxation (C). Inhibition of isomerase activity by CsA results in a loss of the exchange peaks (D). The mixing times used in NMR experiments are indicated.

Figure 2

Quantification of CypA catalysis on CA^N and inhibition by CsA. ¹H/¹H plane of a 3D ¹⁵N-edited NOESY-HSQC shows the diagonal peak of amide G89_trans at 8.0 (¹H) and 109.3 ppm (¹⁵N). (A) An off-diagonal exchange peak between the amide proton of G89 in the trans and cis conformation is evidence for CypA catalysis and its intensity was used to calculate a cis/trans isomerization rate of 10 ± 5 s⁻¹. (B) Addition of excess CsA to sample (A) results in a loss of the exchange peak because of inhibition of isomerase activity. Spectrum B is identical to a spectrum of CA^N alone (data not shown).

Figure 3

Characterization of conformational changes in CA^N upon catalysis and/or binding to CypA. (A) Comparison of ¹H-¹⁵N HSQC-TROSY spectra for CA^N alone (black) and saturated with CypA^WT (green) illustrates that residues remote from the CyPA binding loop are affected by CyPA binding and/or catalysis. (B) The magnitudes of chemical shift changes for the CA^N amides on complex formation with CypA^WT (Δω) are shown as a bar graph. Secondary structure elements and the CypA binding loop (85–93) are labeled. Residues for which Δω is at least 23 Hz, but which could not be unambiguously determined because of signal overlap, are labeled with an asterisk. Chemical shift changes for the side chain NH of Trp 23, 80, 117, and 133 are shown in orange. An expanded view of the G89 region, highlighted in the full spectrum A, is shown in C and D. At saturating concentrations of CypA^WT (CA^N/CypA molar ratio of 1:2), the cis- and trans-bound forms resonate at an average chemical shift because of fast chemical exchange between the two isomers (C). Conversely, both the cis and trans conformers of G89 are observed in the enzyme-bound state when the titration is performed with catalytically inactive CyPA^R55A (D). The chemical shift changes for G89_trans can be followed on titration with CyPA^R55A as shown for ratios of CA^N/CypA^R55A at 1:1 (red), 1:2 (orange), and 1:6 (yellow). However, for G89_cis, the bound form is only observed at higher CyPA^R55A/CA^N ratios in agreement with a higher affinity of the CA^N cis isomer for CypA.

Figure 4

Identification of residues within CA^N that change in chemical shift on binding and catalysis by CypA (22). CypA (blue) is shown bound to the flexible loop between Pro-85 and Pro-93 of CA^N (yellow). Backbone amides of CA^N that shift on CypA binding by more than 14 Hz are highlighted in red. These residues are not only located within the flexible loop (85–100), but also in α4, α5, and α6. Prolines within CA^N (1–146) are shown in green.

Scheme 1

Four-state model of CypA catalysis on CA^N. The capsid protein exists in two free forms with the G80-P90 peptide bond in the trans (CA) and cis conformation (CA). Both forms bind to CypA and are interconverted on the enzyme. The rate of uncatalyzed cis/trans isomerization is slow, whereas the catalyzed rate is fast, resulting in an average chemical shift for the two enzyme-bound forms. The relative affinities of the two isomers of CA^N for CypA determine the cis/trans equilibrium constant on the enzyme, K₄.