Combinatorial optimization of CD4M33
The β-hairpin of CD4M33 (residues 18-27) accounts for about 75% of its interface with HIV-1 gp120 () 24
. A “hotspot” of interaction involves the biphenylalanine at residue 23, which inserts into a gp120 pocket at the interface of several gp120 domains. The biphenyl moiety enhances CD4M33 affinity for gp120 about 10-fold over its phenyl derivative ([Phe23
. To optimize CD4M33, we examined where variability might be best introduced. We avoided altering a number of residues: the six cysteines corresponding to three disulfides, which are vital to the correct miniprotein folding; and residues in the second β-strand of CD4M33, which hydrogen bond to β15-strand of gp120 in a manner similar to the C″-strand of CD4. Instead, we explored the first β-strand of CD4M33, which engages in peripheral interactions with gp120 (), by introducing L-amino acids into positions Lys18
. We also explored the top of the β-hairpin, by introducing L- and D- amino acids into position Gly21
(positions i+1 and i+2 of the turn).
Figure 1 Structure and sequence of CD4M33 bound to gp120. Close-up of the synthetic miniprotein, CD4M33, binding to the HIV-1 gp120 envelope glycoprotein (from pdb accession code 1yyl) 24. Both structure and sequence of CD4M33 are colored yellow, except for residues (more ...)
To enhance solubility and synthesis yield, the biphenylalanine residue at position 23 was replaced by a phenylalanine in the template used for combinatorial synthesis. Cysteines (both L-and D-) were omitted as choices for the four optimized residues to reduce potential misfolding during library synthesis. Iterative deconvolution was used to screen the peptide libraries, so that synthesis was required of only four libraries of, respectively, 19, 19, 34 and 34 sublibraries. Together, the generated variation encompassed 417,316 different peptides.
A combinatorial deconvolution strategy was iteratively used to study the four screened positions at residues 18, 20, 21 and 22. The strategy involved a series of four libraries comprising mixtures in which the identity of an amino acid at a given position was known, and combinations of amino acids were incorporated at the other positions. The overall procedure is illustrated in .
Iterative deconvolution and sublibrary synthesis used in combinatorial optimization of CD4M33.
The first peptide library involved 19 defined (L)-amino acids at position 18, with 19×34×34 variants at positions 20, 21 and 22. Reverse-phase HPLC showed a time shift in retention of the main synthetic peak suggesting proper oxidization of the disulfides. Analyses by MALDI-TOF confirmed the presence of a mass pattern at the desired size. CD spectra were indicative of globally correct folding similar to the CD spectrum of CD4M33 (supplementary Fig. S1
Peptide concentrations were normalized by quantitative amino-acid composition, and each sublibrary of the first library was tested at three different concentrations (10-6
M) for the inhibition of gp120 binding to CD4 by fluorescence anisotropy competition assay 27
() and by ELISA (supplementary Fig. S2
). In the initial optimization, inhibition at 10-8
M was low for both methods. On the other hand, both methods showed that with the sublibraries for position 18 (B1A to B1Y), Arg had the highest activity at 10-6
M, followed by Ala in fluorescence anisotropy and Lys and His in ELISA ( and supplementary Fig. S2
), and position 18 was fixed as an arginine.
Figure 3 Combinatorial optimization of CD4M33: screening of peptide libraries by means of a competitive fluorescence polarization assay. Inhibition of the binding of CD4M33-Fl (1 nM) to gp120HXBc2 (12.5 nM) by libraries B1, B2, B3 and B4 was measured by fluorescence (more ...)
The remaining positions were sequentially identified through an iterative procedure of (re)synthesis and screening (). Thus, a second peptide library was generated that comprised 19 sublibraries of 1156 compounds each (B2A to B2Y), with arginine fixed at position 18 and a defined L-amino acid at position 20. After validation of conformity and assessment of gp120 binding by fluorescence anisotropy () and ELISA (supplementary Fig. S2
), position 20 showed a small (10%) preference for Ala (over Arg and Ser), and position 20 was fixed as an alanine.
A third combinatorial library was synthesized and tested ( and supplementary Figs. S1 and S2
). This library included 34 sublibraries of 34 different miniproteins each (B3A to B3dY) with a fixed amino acid of either L- or D-configuration at position 21. A pronounced difference was observed between sublibraries containing L-amino acids and those containing D-amino acids. A clear preference was observed for residues of D-configuration and to a lesser extent for glycine (which is achiral). The selection at this position for a whole family of amino acids suggested a structural effect of the mutation, which was confirmed by the small difference in activity between the different sublibraries with a D-amino acid. The type II′ β-turn seemed to be the most favourable turn for the activity of our miniproteins. Based on these considerations, position 21 was fixed as a (D)-proline.
The fourth and last library amounted to a parallel synthesis of 34 different peptides with a defined (L) or (D)-amino acid at position 22 (B4A to B4dY). The preferred amino acid here was not as clear as with previous positions. Several amino acids showed roughly equivalent inhibition at the 10-7 M. At lower concentrations, however, a preference was observed for threonine, which was chosen for position 22. The final selected sequence, shown in , contained Arg, Ala, (D)-Pro and Thr at positions 18, 20, 21 and 22, respectively.
Surface-plasmon resonance characterization of CD4-mimetic binding to HIV-1 gp120
The optimized compound and its homologue with a biphenyl instead of the phenyl moiety at position 23 were individually synthesized and named [Phe23]M47 and CD4M47, respectively. Surface-plasmon resonance was utilized to determine the binding parameters between HIV-1 gp120 and the various CD4-mimetic miniproteins as well as 2-domain CD4 (sCD4) ().
Binding parameters of sCD4 and CD4-mimetic miniproteins to gp120
The mimetics or sCD4 were immobilized onto CM5 sensor chips using standard amine coupling, and the kinetic parameters to both HXBc2 and YU2 gp120 were measured with a Biacore 3000. Parameters for HXBc2 and YU2 were similar, though not exactly identical; changes in rates are discussed in the context of HXBc2, with those for YU2 shown in parenthesis when different. The on-rate of CD4M47 was mostly unchanged from the parent CD4M33, about 6-fold (7-fold) faster than the on-rate of sCD4. Optimization appeared to have its greatest effect on the CD4M47 off-rate, which was reduced almost 4-fold from that of CD4M33, to within 3-fold (2-fold) the rate of sCD4. Taken together, these changes in on- and off-rates resulted in an overall Kd for CD4M47 of 2.7 nM (2.1 nM), about 2-fold (4-fold) tighter than that of sCD4.
Kinetic parameters were also measured for the Phe23 variants of the CD4 mimetics. Both [Phe23]M33 and [Phe23]M47 had on-rates about half as fast as their biphenyl counterparts. Off-rates between phenyl- and biphenyl- containing ligands, meanwhile, were similar to within 1.1-fold (1.7-fold). The results suggest that the biphenyl moiety enhances the speed of complex formation. The biphenyl effect on off-rate, meanwhile, is variable, and dependent on strain-specific features.
CD4-mimetic neutralization of virus
To explore how enhancement in affinity translated into virus neutralization, we analyzed the ability of the CD4-mimetic miniproteins to neutralize a diverse panel of envelope clones. We used two assays, a broadly sensitive HIV-entry assay based on JC53BL-13 indicator cells, which is able to give quantified infection within a single cycle of replication 28
and an HIV-1 envelope-mediated fusion assay employing as effectors persistently infected PM1 cells that present the native envelope on their surface 23
CD4 and miniprotein neutralization potencies were evaluated with a diverse range of viral envelopes, ranging from HIV-1 IIIB (which is extremely similar to the HXBc2 envelope used in the optimization procedure) to SIVcpz (which showed a genetic distance of almost 0.5 changes per residue from HXBc2). The neutralization results, expressed as IC50s, are summarized in .
Comparison of antiviral activities between sCD4, CD4M33, [Phe23]M33, CD4M47 and [Phe23]M47
CD4M47 was active over the entire range of diverse isolates, though its potency (IC50 of neutralization) varied substantially from 3 to 370 nM. Only minor enhancements in neutralization potency were observed between CD4M33 and CD4M47 (1.3-4.3-fold over the entire range), less than the observed increase in relative affinity.
Overall, results from neutralization potency agreed with the rank order of measured affinities (Kd), with one notable exception, that of sCD4. Thus, the mimetics showed a rank order of gp120 affinity ([Phe23]M33 > CD4M33 > [Phe23]M47 > CD4M47) that matched the rank order of mimetic neutralization potency. sCD4, however, had an intermediate affinity, but the most potent neutralization. Two possibilities might account for this. First, the larger size of sCD4 (7-times the mass of the miniproteins) might boost its neutralization potency, and second, the contribution of on- and off-rate to neutralization potency is unclear, and the low off-rate of sCD4, slower than any of the mimetics, may enhance its neutralization potency relative to its Kd.
Although CD4M47 displayed only minor improvements in neutralization breadth versus CD4M33, this improvement was seen over the entire range of isolates. Thus, despite increased divergence from the CD4 sequence (Gly to (D)-Pro and Ser to Thr), CD4M47 retained the remarkable breadth of the parent CD4M33 ().
Figure 4 Free energy differences of CD4 and CD4-mimetics as a function of gp120 genetic distance. We previously showed that the difference in free energy (ΔΔG) for two ligands, n=1 and n=2 with 50% inhibitory concentrations of IC50(1) and IC50 (more ...)
Structures of mimetic miniproteins in complex with HIV-1 gp120 core
Both CD4M47 and [Phe23]M47 were crystallized in ternary complexes with core gp120 from the primary HIV-1 isolate YU2 and the antigen binding fragment (Fab) of the 17b antibody. The structures were isomorphous with the previously determined CD4M33 crystal structure 24
and were refined to nominal resolutions of 2.4 and 2.6 Å for the CD4M47 and the [Phe23
]M47-containing complexes, respectively (). Crystal grew as thin plates, and while lower resolution shells were complete, higher resolution shells were less so. Nonetheless, collected data were sufficient to obtain a detailed atomic-level picture of the mimetic-gp120 interactions. Moreover, the crystal lattice contained two independent ternary complexes in the crystal asymmetric unit, allowing the effects of lattice packing and refinement uncertainty to be gauged.
X-ray crystallographic data and refinement statistics
The structure of the newly determined CD4M47 complex is shown in (along with the structure of the [Phe23]M47 variant). Superpositions with CD4M33 are shown in and with CD4 in . The mimetic structures, before and after combinatorial optimization, were extremely similar, with root-mean-square (rms) deviations for the entire backbone of 0.27-0.67 Å, and of 0.19-0.26 Å for residues 18-22. Residue 18 was not closely constrained by gp120, with considerable distance between this portion of the mimetic and gp120. The added bulk of Arg 18 in CD4M47 versus the parent CD4M33 Lys allowed for additional contact surface area with gp120, although these contacts were somewhat tenuous. Residues 20 and 21 were more constrained by gp120; optimization added a total of four carbon atoms to these two residues, increasing the fit between mimetic and gp120 ().
Figure 5 Crystal structures of CD4M47 and [Phe23]M47 in complex with YU2 core gp120 and Fab 17b. (A) Overall view of gp120 (red), CD4M47 (teal), and Fab 17b (gray). The orientation shown is 90° about a vertical axis from the orientations in C-H, and the (more ...)
One measure of the degree of fit between two molecules is their surface complementarity 29
. The surface complementarity between CD4 and core gp120 ranged from 0.674 for HXBc2 core gp120 and 0.716 for the YU2 core (with the value for an optimal fit being 1.000). With the CD4-mimetic miniproteins, surface complementarities ranged from 0.769 for both lattice molecules of CD4M33 to 0.801 for molecule 1 of [Phe23
]M33 (supplementary Table S1
). Within this range, differences from lattice variation had a considerable effect, with the two molecules of CD4M47 showing surface complementarities of 0.769 and 0.793. Perhaps because of the highly restricted range, no correlation was observed between surface complementary and overall affinity.
A second way to analyze structural interfaces is to quantify the molecular contact surface. For mimetic residues 18, 20 and 21, in each case, optimization resulted in an increase in the contact surface. For residue 18, the increase was roughly 20Å2, and for residues 21 and 22, the increase was roughly 10Å2 (). Thus, the total increase in contact surface from the optimized residues was ~40Å2, or about 10% in the total mimetic contact surface.
Contact surface area of gp120 by CD4 and or CD4-mimetics*
While these results suggest a correlation between affinity for gp120 and contact surface area between mimetic and gp120, they must be tempered by the caveat that other amino acids that might increase surface area were not observed to enhance affinity in the optimization. Thus, while interactive surface area was observed to correlate with enhanced affinity, other factors such as surface complementarity and the specific chemistry of the interactions need to be taken into account.
Structural mimicry induced in gp120 by miniproteins and correlation with induced affinity for CCR5 coreceptor
Another structural measure of the interaction between two molecules is the similarity in their induced conformational change. In the case of the gp120-CD4 interaction (and also the gp120-CD4-mimetic interaction 24
), gp120 undergoes substantial structural rearrangement, with much of the secondary structure of the gp120-inner domain rearranging 30-32
We used distance-sorted distance matrices 24, 33
to quantify conformational comparisons of gp120 around the interaction hotspot (). In the 10Å shell extending from the Cα of residue 23 on the mimetic, the structures of the gp120 molecules complexed to CD4M47 showed normalized linear difference distances of 0.22-0.23Å, similar to that seen with CD4M33 (0.22-0.24Å). The linear difference distances decreased with [Phe23
]M47, with normalized linear difference distances of 0.16-0.20Å, a bit larger than with the [Phe23
]M33 analogue (0.13Å). Thus, in terms of induced gp120 structure, [Phe23
]M33 remained the closest mimic to CD4, with [Phe23
]M47 intermediate to CD4M47 and CD4M33.
Figure 6 Distance-sorted difference distance matrix analysis of induced gp120 conformation. Difference distance matrices provide an alignment-independent means to assess structural similarity 33, 62, and we previously showed that this type of analysis could be (more ...)
Here the two independent copies of the mimetic in the P21 lattice provide a means to verify the relevance of the distance-sorted difference distance metrics. However, the overall linear and square difference distances were small, only a bit larger than coordinate error. Moreover, the structural measurements were actually made in the ternary complex of mimetic, gp120 and 17b, and thus the gp120 conformation was influenced by 17b. We therefore sought to determine whether these small matrix differences were biologically relevant.
Unliganded gp120 binds poorly to the CCR5 coreceptor, with high affinity CCR5-gp120 interaction requiring a CD4-induced conformational change 34
. To define the biological effects of the mimetic-induced changes in gp120, we compared the amount of gp120 bound to CCR5+
cell lines in the absence or in the presence of CD4 or of one of the CD4-mimetic miniproteins. The amount of gp120 bound to CCR5+
cell lines was evaluated with two CCR5-tropic gp120 (SF162 and YU2, the same as used for co-crystals). Sequential incubations were carried out with the gp120-reactive antibody, D7324, and with a secondary PE-conjugated antibody. As shown in , [Phe23
]M33 and [Phe23
]M47 were more capable than the biphenylalanine derivatives in inducing a CCR5-binding conformation. Moreover, the induced affinity of gp120 to CCR5 correlated well with induced structural mimicry, as quantified by distance-sorted distance matrices.
Figure 7 gp120-induced affinity for CCR5 and correlation with structural mimicry. The binding of recombinant gp120 envelope glycoprotein to CCR5+-CHO cells in absence or in presence of saturating concentrations of sCD4 or CD4-mimetic miniprotein was analyzed by (more ...)