Recently we determined that the N-terminal binding region of Vpr to CypA is comprised of the heptapeptide 32
centered at Pro-35 [14
]. Mutation of the central Pro residue (P35A) causes a loss of binding through disruption of the Pro-dependent N-terminal heptapeptide binding region. In the context of the full length molecule this mutation also results in merging of helix 1 and 2, which in turn causes a substantial change in the hydrophobic core of the protein as the anti-parallel folded structure of the N-terminal and central helices is no longer possible [38
In this paper we have characterized a C-terminal region of Vpr, comprising the 16 residues 75
, with high binding affinity for CypA. This region of Vpr does not contain any Pro residues, but binds much more strongly to CypA than to the N-terminal binding region (Figure ). The fact that the mutant peptide Vpr75-90
R80A binds considerably weaker to CypA than the wt
peptide (Figure ) confirmed that Arg-80 is a key residue in the C-terminal binding region. Arg-80 is considered to be located in proximity to, but not included in, the well-defined helix 3 (residues 55-77) of the structured protein formed under membranous conditions at physiological pH [45
]. The secondary structure NOEs of residues 78-90 of full-length Vpr found by Morellet et al. [45
] are mainly restricted to those between NH-NH(i, i+1)
, in addition to a few Hα-NH(i, i+2)
and Hα-NH(i, i+3)
, indicating that these residues comprise a region with a relatively weak α-helical structure. Indeed, our NMR data of wt
R80A confirmed this interpretation and revealed that the propensity for weak helical structure of the C-terminal binding region is essentially unaffected by the mutation of Arg-80 to Ala (Figure ). Thus, the mutation prevents binding of Vpr to CypA through a local change in the C-terminal binding region rather than any change in the tertiary structure of Vpr.
In aqueous solution Vpr is present as high order aggregates (~decamers) with a lower percentage of higher multimers [8
]. According to Fritz et al. oligomerization is mediated by the Vpr hydrophobic core but not by the flexible N- and C-terminal domains [47
]. The C-terminal binding domain of Vpr to CypA is located beyond the residues that have been shown experimentally to be involved in oligomerization of Vpr. In agreement with this we used the statistical mechanics algorithm TANGO, which identifies aggregation-prone regions of peptides and denatured proteins using a set of balanced physico-chemical parameters [48
]. According to the TANGO algorithm, a score of ≤ 0.02% indicates no aggregation, 0.02-5.0% indicates moderate aggregation, and ≥ 5.0% indicates high aggregation propensities. Application of this program predicted a region of 16 residues (Ala-55 to Ile-70) populating the oligomerization state to more than 5% per residue (5.09 - 35.56% per residue). Four further residues were predicted with much lower score, namely Thr-53 (1.30%), Trp-54 (3.50%), His-71 (0.84%) and Phe-72 (0.80%). The fact that the dissociation constant of full-length Vpr is in the same order of magnitude as the dissociation constant of the C-terminal binding domain Vpr75-90
does not indicate that oligomerization of Vpr influences the interaction with CypA significantly.
Previous studies have shown that the mutants Vpr P35A and Vpr R80A were key residues for coimmunoprecipitation of Vpr and CypA, and when replaced abrogated the coimmunoprecipitation to CypA [12
]. Although, these authors concluded that residues beyond Vpr1-40
also are important for binding to CypA, no further explanation was provided. Our data independently confirm the importance of these residues for the interactions and indicate that the C-terminal binding is quantitatively stronger than the N-terminal binding to CypA (Figure ). However, the fact that the decapeptide Vpr75-84
fails to bind to CypA (Figure ) clearly demonstrates that Arg-80 and its nearby surrounding residues are insufficient for the interaction to occur. Reduced binding affinity was also observed for a mutant Vpr75-90
peptide where Arg-80 was conserved, demonstrating the importance of an intact 16 residue C-terminal binding domain of Vpr.
A simultaneous interaction of both Vpr sites with CypA was suggested as SPR sensorgrams of full-length Vpr interacting with immobilized CypA gave an optimal fit with a bivalent analyte model (Figure ). Taken together, these data indicate that a simultaneous binding of the N- and C-terminal domains is required for full-length Vpr to interact with CypA. The dissociation constant (KD
) of full-length Vpr was found to be approximately 0.32 μM (Table ), which implies that the Vpr-CypA interaction may be stronger than the well characterized interaction of HIV-1 CA with CypA, which has been determined previously by SPR spectroscopy (KD
16 ± 4 μM) [50
]. This suggests that the CypA-Vpr interaction has a functional role in the relationship between the host and pathogen.
The access to detailed information of the interaction of full-length Vpr with CypA, based on the experimental characterization of the N- and C-terminal binding domains of Vpr, now allows us to visualize the interaction of full-length Vpr with CypA. As prerequisites, we have used the NMR structure of Vpr [45
] and X-ray structure of CypA [51
] as rigid units, together with knowledge of the key residues of the Pro-dependent and Pro-independent binding domains of CypA [39
]. A cartoon illustrating the Vpr-CypA complex based on these parameters was generated with the ZDOCK algorithm [52
] and is shown in Figure . This crude model suggests that a folded Vpr structure is required to provide N- and C-terminal binding regions sufficiently close in space in the Vpr-CypA complex (Figure ) for the cooperative interaction with the two binding sites of CypA. Currently one should keep in mind that the model has its limitations but clearly rationalizes the findings presented here. The structure of Vpr used, which is the potentially variable/flexible component of the model, is the limiting structure at low pH in 30% aqueous acetonitrile [45
]. However, a more flexible structure of Vpr, which would be expected under the hydrophilic conditions at physiological pH [8
] used in the Biacore experiments performed to characterize the interaction, would be beneficial for improving the model and, thus, provide more accurate details of the interactions of the Vpr-CypA complex at the atomic level.
Figure 7 Cartoon of the complex of Vpr and CypA. The cartoon shows the 30 KDa complex of CypA with full-length Vpr where selected residues of the cooperative N- and C-terminal binding domains of Vpr and the two binding sites of CypA suggested by Demange et al. (more ...)
Most residues of the N- and C-terminal binding domains of Vpr are highly conserved among HIV-1 strains (with the exception of Ile-37 of the N-terminal binding domain and Arg-77, Val-83, Thr-84, Arg-85, Gln-86 and Ala-89 of the C-terminal binding [54
]), indicating that maintenance of these structural regions of the protein is important for the viral life cycle.
The N-terminal Vpr binding domain is located between helix 1 and 2, and the C-terminal Vpr binding domain is located in an arginine-rich region in proximity to the third α-helical domain of Vpr (Figure ). Although the biological importance of the binding of Vpr to CypA remains elusive, multiple functions of Vpr are connected to the two regions of the protein which bind cooperatively to CypA, including apoptosis [55
], reverse transcriptase (RT) activity [36
], replication of R5 tropic HIV-1 [38
], nuclear localization of the protein [30
], G2 cell cycle arrest [30
] and binding of Vpr to DNA and RNA, which is linked to the ability of activating the ATR (ataxia-telangiectasia and Rad3-related) pathway leading to G2 arrest [72
] (Figure ). The importance of both domains to achieve binding of full-length Vpr to CypA suggests that the biological relevance of the interaction may be associated with other functional interactions of Vpr involving both the N- and C-terminal regions (Figure ). Previous studies have presented contradictory results regarding the possibility that the interaction of Vpr with CypA is involved in induction of G2 arrest or that it has any effect on Vpr expression [12
]. However, other studies have independently shown that Vpr induced G2 arrest is dependent on residues belonging to both the N- and C-terminal binding regions of Vpr to CypA [30
]. Residues within the C-terminal domain of Vpr including Arg-80, which was found essential for retaining a strong binding to CypA in this study, are also considered to be important for increasing RT activity [36
] and Vpr induced apoptosis [56
]. Furthermore, residues in the N-terminal binding region of Vpr are important for Vpr induced apoptosis (Figure ) [57
], indicating that, both the N- and C-terminal CypA binding domains are involved in this function of Vpr. Moreover, the nuclear localization of Vpr and replication of R5 tropic HIV-1 are related to residues included in both N- and C-terminal CypA binding domains of Vpr (Figure ). The identification of N- and C-terminal binding domains of Vpr, which cooperatively bind to CypA (Figure ), should encourage further investigations into the biological relevance of the interaction of Vpr with the host cellular factor CypA, as it is likely that this strong interaction is of importance in the viral life cycle of HIV-1.
Figure 8 Previously determined biological functions associated with the N- and C-terminal binding domains of Vpr to CypA. Linear structure of full length Vpr with incorporated biological functions connected to the determined N- and C-terminal binding regions of (more ...)