The intriguing Cyclophilin A-HIV-1 Vpr interaction: prolyl cis/trans isomerisation catalysis and specific binding

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.


Background
The 96 amino acid virion-associated multifunctional viral protein R (Vpr) [1,2] is encoded by primate lentiviruses, the human immunodeficiency viruses, types 1 and 2 (HIV-1/HIV-2), and simian immunodeficiency viruses (SIV). This accessory protein fulfils multiple functions in the viral life cycle including increase of viral replication in non-dividing host cells, induction of G2 cell-cycle arrest [3,4], apoptosis [5,6], and transduction through cell membranes [7]. Vpr facilitates transport of the pre-integration complex into the nucleus of non-dividing cells [8] and interacts with several cellular factors, including the human peptidyl prolyl isomerase CypA [9].
The interaction of HIV-1 Vpr with CypA is known to occur in vitro and in vivo [9][10][11]. In addition to the extensively studied interaction between CypA and HIV-1 capsid, that is crucial for viral replication [12,13], CypA was also reported to be significant for the de novo synthesis of Vpr, as the Vpr-mediated cell cycle arrest in HIV-1 infected T cells appeared to be eliminated in the absence of CypA activity [9]. However, more recently Ardon et al. [10] concluded that the interaction of Vpr with CypA is independent of the ability of Vpr to induce cell cycle arrest. Nonetheless, specific inhibitors of the prolyl cis/trans isomerase activity of CypA, such as cyclosporine A and SDZ-NIM811 inhibit HIV replication [11,[14][15][16][17][18].
Previous structural studies of Vpr by 2D NMR spectroscopy in aqueous organic solvents provided evidence of proline cis/trans isomerism for the highly conserved N-terminal Pro-5, -10, -14 and -35 of Vpr [19]. In particular Pro-35 exhibited a relatively high proportion of the cis isomer under these solvent conditions (15% cis isomer content). This suggested prolyl cis/trans isomerisation may be important for the folding of the molecule. At pH 7 Vpr has a relatively random structure in aqueous solution but assumes a folded structure in a hydrophobic membranous environment [7,19]. This fact together with observation of considerable amounts of CypA in virions [20] prompted a study, using surface plasmon resonance (SPR) spectroscopy, of the interaction of Vpr with the prolyl cis/trans isomerase CypA [9]. A qualitative interaction was detected for N-terminal peptides containing Pro-35, indicating an essential role for this Pro residue, although this could not be quantified. Indeed the interaction of Vpr with CypA could not be confirmed under the solution conditions used. Hence, the nature of the interaction of CypA with Pro-35 of N-terminal Vpr has remained undefined.
The catalytic activity of CypA, as a peptidyl-prolyl cis/ trans isomerase (PPiase), has previously been studied on short model peptides containing one Pro residue. Thus, based on interaction studies of eight short model peptides, each comprising four residues containing only one Pro residue preceded by a variety of different amino acids, Harrison and Stein [21] concluded that CypA exhibited a broad tolerance as a prolyl cis/trans isomerase of several substrates. Endrich et al. [22] reported that CypA interacts with three 14-37 residues HIV-1 Capsid-derived peptides, each containing 1-4 Pro residues and at least one Gly-Pro motif. However, exact information regarding the interaction of individual Pro residues within peptides containing more than one Pro unit was not accessible with the applied methodology.
The suitability of NMR spectroscopy for studying the catalytic activity of CypA has been demonstrated in a more limited number of reports. NMR spectroscopy is the only method wherefrom information about prolyl cis/trans isomerase interaction of CypA with individual Pro residues of a peptide, containing several Pro residues, is accessible at atomic resolution. However when determined, only selective interactions involving one of the Pro residues in the peptides [12,23] or the full length protein [13] containing more than one Pro residue have been reported.

Results
The combination and access to both highly pure recombinant CypA and synthetic N-terminal Vpr peptides, as well as sensitive NMR and SPR facilities, has allowed us to probe the interactions of Vpr and CypA in more detail than previously.
The N-terminal region of Vpr includes four highly conserved Pro residues at positions 5, 10, 14 and 35, respectively ( Fig. 1) (more than 98% conserved according to references [19] and [26]). Among these, the presence of Pro-35 is vital for several functional interactions of Vpr. The tertiary structure of Vpr observed in aqueous and 30% aqueous acetonitrile solution at low pH is characterized by a hydrophobic core formed by the three α-helices [25] and partly reliant on the presence of the Pro-35 residue that acts as a helix breaker between helix 1 comprising residues 17-33, in which the p6 binding domain of Vpr is located [27], and helix 2 (comprising residues 38-50) of Vpr [28]. At pH 7 Vpr is essentially unstructured in aqueous solution and requires under these conditions a hydrophobic environment to achieve α-helical structure [7,19] (Additional file 1, Fig. S1).
It is particularly noteworthy that exchange peaks between analogous 1 H signals of the cis and trans isomer were not observed in the NOESY spectra of any of the pure peptides, which is a clear indication that only a slow cis/trans interconversion on the NMR time scale is taking place in the absence of enzymatic catalysis. As a consequence of the relatively large energy barrier, uncatalyzed isomerisation is a rather slow process with an interconversion time on the order of several minutes [29,30].
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 1 H chemical shifts of sVpr 1-20 were recorded, followed by addition of catalytic amounts of CypA (molar ratio sVpr 1-20 -CypA 224:1; molar ratio sVpr 1-20 proline substrate-CypA 672:1). After addition of catalytic amounts of CypA, particularly the HN-HN region of the 2D 1 H-1 H NOESY NMR spectrum of sVpr 1-20 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 sVpr 21-40 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.
Thus we have direct experimental evidence that all four conserved Pro residues in Vpr undergo cis/trans isomerism in aqueous solution at pH 7 that is catalyzed by CypA. Only small amounts of enzyme are required and the NMR method is sufficiently sensitive to detect these effects in ratios of substrate to enzyme as high as 672:1. The applied relative proportions of enzyme to substrate were considerably lower than the relative proportions of CypA (enzyme to substrate 1:8-1:300) used in previous studies with NMR techniques [12,13,23,[31][32][33][34][35].
Kinetic analysis of the Biacore binding data showed deviations from a pseudo-first order 1:1 binding model. The binding curves of CypA with sVpr 25-40 , sVpr 21-40 , sVpr 1-40 and the short peptides sVpr 30-40 and sVpr [32][33][34][35][36][37][38] were best described by a two-state binding interaction model (Fig. 7, Additional file 1, Fig. S3). The minor deviation observed between the experimental and modelled curve for sVpr 21-40 at the highest concentration, i. e. 400 μM, may be due to aggregation of the peptide at this concentration. The two-state binding model, also called the conformational change model, is based on a 1:1 binding of an analyte to the immobilized ligand followed by a conformational change in the complex. This model should be regarded as indicative, rather than as direct evidence, for a conformational change upon binding [38]. However, our NMR data confirm that CypA acts as a prolyl cis/trans isomerase of the highly conserved Pro residues of N-terminal Vpr including Pro-35, hence a conformational change upon binding is probable. Thus, CypA catalyzes the prolyl cis/trans interconversion during binding to the sVpr peptides. The interconversion rate constant derived from the ROESY NMR experiments was determined to be 0.19 ± 0.01 s -1 . Thus, a peptide-CypA complex would undergo approximately 12 prolyl cis/trans interconversions during the 60 sec. association phase of the Biacore experiment. From the two-state binding model the association rate (k a1 ), dissociation rate (k d1 ), forward rate (k a2 ) and backward  40 P35N) (F) were tested for binding to immobilized recombinant CypA using a SPR biosensor system. A, B and C were injected at concentrations ranging from 0-400 μM over CM5 chip immobilized with 918 RU CypA, while D, E and F were injected at concentrations ranging from 0-200 μM. The curves A, B, C, D and E were best fit to a two state reaction model (Fig. 7). k a1 , k a2 , k d1 , k d2 and K D were calculated for respective sensograms (Table 1). rate (k d2 ) constants were calculated, and subsequently the magnitude of the dissociation constants (K D ) could be calculated (1/((k a1 /k d1 )*(k a2 /k d2 )) ( Table 1).
The two facts, that sVpr 1-20 did not bind to CypA in the Biacore assay (Fig. 6) although CypA was shown by our NMR studies to interact with these Pro residues of the peptide as a prolyl cis/trans isomerase (Fig. 3, 5) and that the presence of Pro-35 on the other hand is required for binding, indicate the observed specific binding of N-terminal Vpr peptides to CypA require a binding region that is structurally dependent on Pro-35.
To identify and determine the specific binding region of N-terminal Vpr to CypA, SPR studies of the shorter Vpr peptides sVpr 30-40 , sVpr 32-38 and sVpr 33-37 were performed. The sensograms revealed that sVpr 30-40 and sVpr 32-38 maintain the strong binding to CypA similar to that of longer N-terminal Vpr peptides (Fig. 8). In contrast, the shortest peptide sVpr 33-37 binds considerably weaker. The remarkably weaker response of the binding curves of sVpr [33][34][35][36][37] to CypA compared with the longer peptides, demonstrates that the shortest peptide sequence maintaining strong binding is the heptapeptide sVpr 32-38 . 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.

Discussion
As a continuation of our previous discovery of the interaction of CypA with Vpr, these interactions have in this work been characterized in detail at atomic resolution. Direct experimental evidence that all four conserved Pro residues in Vpr undergo cis/trans isomerism in aqueous solution at pH 7 that is catalyzed by CypA was achieved. Only small amounts of enzyme are required and the NMR method is sufficiently sensitive to detect these effects in ratios of substrate to enzyme as high as 672:1.
The apparent differentiation between the results originating from interaction studies performed by NMR spectroscopy and SPR indicates a different additional mode of interaction observed in the latter case. The fact that only N-terminal Vpr peptides containing Pro-35 bind to CypA in the Biacore assay and strong binding is maintained in the heptapeptide sVpr 32-38 , although CypA also catalyzes prolyl cis/trans interconversions of Pro-5, 10 and 14 of sVpr 1-20 as shown by NMR spectroscopy at atomic resolution, is convincing evidence that CypA only binds specifically to peptides containing the seven-residue motif RHFPRIW centred at Pro-35 of Nterminal Vpr. With the exception of Ile-37, residues comprising the binding region of N-terminal Vpr to CypA are highly conserved [26]. This region includes the loop region connecting helices 1 and 2 of Vpr, in addition to Arg-32 and His-33 that terminates the welldefined helix 1 of Vpr.
The situation found here is analogous to the NMR study reported by Bosco et al. [12] where CypA not only interacts as a chaperone with Pro-90 of N-terminal HIV-1 Capsid but also selectively catalyzes the prolyl cis/trans interconversion of the Gly-89 -Pro-90 bond. Crystal structure of the complex of CypA with the Nterminal HIV-1 Capsid peptide (residues 1-151) revealed that the binding region of HIV-1 Capsid to CypA encompasses the nine residues 85 to 93 (PVHAGPIAP) belonging to a loop region connecting helices 4 and 5, respectively, of HIV-1 Capsid [39]. Based on X-ray crystallography, Vajdos et al. [40] reported that the HIV-1 Capsid-derived hexapeptide HAGP 90 IA maintained the strong interaction with CypA, thus limiting the binding region of HIV-1 Capsid to include these six residues. Piotukh et al. [41] used phage display to delineate the requirements for stable binding of CypA to linear peptide motifs containing the consensus motif FGPXLp that is present in many human proteins. This binding motif is quite different from the sequence RHFPRIW found here that defines the binding region of N-terminal Vpr to CypA and also differs from the binding region of HIV-1 Capsid (HAGPIA), indicating that the Pro-dependent binding pocket of CypA is tolerant of substrates with several different sequences.
Pro-35 in Vpr is located in a loop region separating helix 1 and helix 2 [25]. Structural data for an N-terminal mutant of Vpr in which Pro-35 is exchanged for Asn indicate a merge of helices 1 and 2 leading to a significant conformational change in the hydrophobic core of Vpr [11]. This will thus disrupt the hydrophobic interactions of helices 1 and 2 with helix 3 that were observed by long-range NOEs [25]. The tertiary structure of wt Vpr consists of three well-defined α-helices consisting of residues 17-33 (helix 1), 38-50 (helix 2) and 56-77 (helix 3), respectively, folded around a hydrophobic core constituted of Leu, Ile, Val and aromatic residues [25]. According to Morellet et al. [25], the hydrophilic accessible domains of Vpr exposed to the solvent molecules comprise the acidic residues Glu-21, Glu-22, Glu-24, Glu-25, Asn-28 and Glu-29 of helix 1, the basic residues Arg-62, Arg-73, Arg-77, Gln-65 and Gln-66 of helix 3 and the basic residues Arg-80, Arg-87, Arg-88, Arg-90 and Lys-95 of the flexible C-terminal domain, respectively. The fact that the mutant Vpr R80A failed to co-immunoprecipitate with CypA [10] may indicate that the binding of full-length Vpr to CypA is also dependent on the maintenance of the tertiary structure of Vpr. Clearly there are at least two highly conserved residues in the molecule that are crucial for maintaining the interaction of CypA with Vpr, namely Pro-35 and Arg-80.
Previous literature has indicated that CypA is relatively tolerant as a prolyl cis/trans isomerase to a variety of different substrates [21]; however, these studies have been performed exclusively on short peptides comprised of four amino acids and containing only one Pro residue. To our knowledge catalysis by CypA of prolyl cis/  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 k a1 , k a2 , k d1 , k d2 and K D were calculated for the respective sensograms (Table 1.).
trans interconversions of more than one Pro residue in a peptide containing several Pro residues has not previously been reported. However, exclusively selective prolyl cis/trans isomerase catalysis mediated by CypA on only one Pro unit have been reported for peptides containing more than one Pro residue [12,13,23].
The binding curves from Biacore analyses of the specific interaction of Pro-35 were consistent with a twostate binding interaction model involving a conformational change during the binding. This is in accord with the catalysis of prolyl cis/trans isomerism observed by NMR in solution, indicating that the RHFPRIW motif of N-terminal Vpr binds the catalytic site of CypA, which maintains the ability to catalyze the prolyl cis/trans interconversion during binding.
The fact that CypA specifically binds to the inter-helical loop region included in the RHFPRIW binding motif centred at Pro-35, under solution conditions where Vpr has a relatively random structure, suggests a mechanism in which CypA may act as a folding chaperone. Whether this plays a role in the de novo synthesis of Vpr, in which CypA appears to be involved, remains to be determined [9]. Clearly the functional consequences of the interactions of CypA still remain to be determined. Further studies will necessitate the evaluation of these interactions in the context of the full length Vpr molecule in the various environments that it encounters in vivo.

Conclusions
Binding of CypA to a defined inter-helical loop region of HIV-1 Vpr is highly specific, which is analogous to the previously characterised HIV-1 Capsid-CypA interaction. The loop region centred at Pro-35 of Vpr comprising the binding motif to CypA maintains the integrity of the N-terminal and middle helices of Vpr and is functionally important for incorporation of Vpr into virus particles and replication of R5-tropic HIV-1 in human lymphoid tissue [28]. CypA acts as a catalyst of prolyl cis/trans interconversions of Pro 5, 10, 14 and 35 of N-terminal HIV-1 Vpr, which may indicate a folding chaperone role of CypA in HIV replication compatible with the fact that inhibitors of CypA, which inhibit CypA-Vpr binding [9,10] and CypA-mediated prolyl cis/trans catalysis, also suppress HIV-1 replication in cell culture.

Cyclosporine A and CypA
Cyclosporine A was purchased from Sigma (C3662; CAS number 59865-13-3). The procedure on which the purification of recombinant human CypA is based has previously been described by others [42]. The E. coli strain BL21(DE3) (Invitrogen) was transformed with an pET11c expression vector encoding human CypA [43]. A 4-liter culture of E. coli BL21(DE3) was grown at 37°C in a LB medium containing ampicillin to an A 595 of 0.8 and isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM. The incubation was continued for additional 8 h. The cells were harvested (3000 g, 15 min), washed with Tris buffer (1 mM DTT, 20 mM TrisHCl, pH 7, 8) and 12 g cell pellet was resuspended in 150 ml of the same buffer. Lysis was performed on ice using a BL12 sonifier (Branson). After separation of the cell debris by centrifugation (48000 g, 15 min) nucleic acids were precipitated by protamine sulphate (0.4% final concentration). The crude cell extract was dialyzed against the Tris buffer overnight and loaded onto a DEAE-Sepharose column (HiPrep16/ 10, GE Healthcare) equilibrated with 4 column volumes (CV) of Tris-buffer and the flow-through fractions were collected, containing essentially pure CypA. These fractions were combined and dialyzed against a 30 mM MES buffer pH 6.0 overnight. For final purification and concentration of CypA, the dialyzed solution was loaded on a MonoS cation exchange column (5/50 GL, GE Healthcare) which was equilibrated with 4 CV of the MES buffer. The final elution step of the bound CypA was performed with a 0-1 M NaCl gradient and yielded 30 mg of highly concentrated and pure CypA fractions, which were analysed by Coomassie staining and Maldi MS to check the purity and the correct molecular weight of the protein (Additional file 1, Fig. S4).

Mass Spectrometry
Matrix assisted laser desorption ionization mass spectra (MALDI-MS) were recorded on a Voyager-DE PRO BioSpectrometry Workstation from Applied Biosystems. Samples were dissolved in 50% aqueous acetonitrile and a-cyano-4-hydroxycinnamic acid was used as matrix. Positive ion electrospray ionization mass spectra (ESI-MS) were recorded on a micromass Q-Tof-2 mass spectrometer. Samples were dissolved in 70% aqueous methanol and infused into the electrospray chamber with a needle voltage of 0.9 kV at a flow rate of 40 nl/min.

Nuclear Magnetic Resonance (NMR) Spectroscopy
2D 1 H Total correlation spectroscopy (TOCSY), Nuclear Overhauser enhancement spectroscopy (NOESY) and Rotating frame Overhauser enhancement spectroscopy (ROESY) NMR experiments were performed at 600. 13 MHz on a Bruker Avance 600 MHz instrument equipped with an UltraShield Plus magnet and a triple resonance cryoprobe with gradient unit. Individual samples were dissolved in 600 μl 50 mM aqueous phosphate buffer pH 7.0 containing 10% D 2 O (v/v), at concentrations between 1-2 mM. The 2D NMR experiments were performed at 300 K without spinning with mixing times of 110 ms for the TOCSY experiments, 250 ms for the NOESY experiments and 500 ms for the ROESY experiments, respectively. Efficient suppression of the water signal was achieved with application of excitation sculpting in the 1 D 1 H and the 2D 1 H TOCSY and NOESY NMR experiments [44] and presat in the 2D 1 H ROESY experiments [45]. 1 H signal assignments of the NMR spectra were achieved by identification of the individual spin systems in the 2D 1 H TOCSY spectra, combined with observations of sequence-specific short-distance crosspeaks (Hα-HN i, i+1) in the 2D 1 H-1 H NOESY spectra [9,46]. Readily recognisable spin systems were used as starting points for correlation of the individual spin systems observed in the TOCSY and NOESY spectra with individual residues in the peptide sequences. Acquisition of data, processing and spectral analysis were performed with Bruker Topspin 1.3 software. Assigned NMR data of the N-terminal Vpr peptides have been deposited at the BMRB (Accession number 17003).

Determination of rate constant for Pro cis/trans interconversion by ROESY NMR
A series of ROESY experiments of Vpr 32-38 (sample concentration 4.2 mM) with addition of 50 μl 0.11 mM CypA (sample concentration of CypA 8.5 μM; total volume 650 μl); relative proportions of substrate to enzyme 494:1, maintaining the same experimental condition as described above but with variable mixing time (500 ms, 450 ms, 400 ms, 350 ms, 300 ms, 250 ms 200 ms 150 ms, 100 ms and 50 ms, respectively) were performed. Volume integration of exchange peaks were carried out with Sparky software [47] and rate constants for each mixing time were calculated as described in Keller et al. [48].

by addition of cyclosporine A
The catalytic prolyl cis/trans isomerase interactions of CypA with sVpr 1-20 and sVpr 21-40 was inhibited by addition of excessive amounts of cyclosporine A dissolved in 5 μl deuterated dimethylsulfoxide (DMSO-d6). Addition of 6 μl pure DMSO did not inhibit the catalytic prolyl cis/trans isomerase interactions of CypA with a model peptide showing that CypA keeps its enzyme activity in 1% DMSO solution. The disappearance of NMR exchange peaks, originating from the catalytic prolyl cis/ trans isomerase interactions of CypA with sVpr 1-20 and sVpr 21-40 , after addition of cyclosporine A was revealed by supposition of analogous NOESY and ROESY spectra prior to and after addition of cyclosporine A, using Bruker Topspin 1.3 software.

Biacore Spectroscopy
SPR measurements were performed at 25°C on a Biacore T100 instrument (Biacore AB, Uppsala, Sweden) equipped with a CM5 research-grade sensor chip. CypA was immobilized in FC 2 and FC 4 to 180 and 918 RU (response units) respectively, using standard amine-coupling chemistry in two flow cells. FC 1 and FC 3 were treated according to FC 2 and FC 4 except for CypA immobilisation and functioned as reference cells. The synthetic Vpr peptides sVpr 1-20 , sVpr 21-40 , sVpr 25-40 , sVpr 30-40 , sVpr 32-38 and sVpr 33-37 were dissolved at four concentrations ranging from 50 to 400 μM in the running buffer (HBS-P buffer pH 7.4). Due to restricted solubility sVpr 1-40 , sVpr 1-40 P35N and sVpr 1-40 P5,10,14N were dissolved at lower concentrations ranging from 25-200 μM in the same buffer. The samples were injected over the flow cells at a flow rate of 30 μl/min. Data were collected at 2.5 Hz during the 60 s association and 120 s dissociation phase, and were automatically corrected for bulk buffer effects and unspecific binding of Vpr peptides to the chip matrix.

Analysis of biosensor data
Affinity, association and dissociation rate constants were obtained from sensograms by the Biacore T100 evaluation software version 2.0.1 in accordance with the global curve fit model. Sensorgram data for the four different concentrations were fitted to several binding models including 1:1 (Langmuir) binding model (A+B ↔ AB), two-state reaction (conformational change) model (A+B ↔ AB ↔ AB*) and heterogeneous ligand model (interaction one: A+B1 ↔ AB1; interaction two: A+B2 ↔ AB2). Kinetic constants were calculated for the best fitted model.