Selection of SMM chemokines and CXCR4 mutants for binding site mapping experiments.
To identify novel ligand functional sites on CXCR4 with our chemical biology approach, the following SMM chemokines were chosen for the present study: (1-10)-vMIP-II-(9-68)-SDF-1α, d(1-8)-SDF-1α, d(1-10)-vMIP-II-(9-68)-SDF-1α, and d(1-10)-vMIP-II, all of which contain amino acid substitutions and/or d-amino-acid replacement at the N terminus. Natural chemokines, SDF-1α and vMIP-II, were used as positive controls and for comparisons. Their sequences and modifications are provided in Table . There are two different types of d-amino-acid-containing SMM chemokines investigated in this study, which were based on vMIP-II and SDF-1α, respectively. d(1-10)-vMIP-II was derived from vMIP-II by replacing the N-terminal (1-10) sequence module of vMIP-II with d-amino acids, whereas d(1-8)-SDF-1α and d(1-10)-vMIP-II-(9-68)-SDF-1α were based on SDF-1α and contain the replacement of the N-terminal (1-8) residues of SDF-1α by d-amino acids or all dforms of the N-terminal (1-10) residues of vMIP-II, respectively. Such d-amino-acid-containing SMM chemokines were chosen as probes to study their binding sites because of their improved CXCR4 selectivity, binding affinities, and/or anti-HIV activities (unpublished data).
List of SMM chemokines and their sequences and modifications
As for the CXCR4 mutants, with the exception of DNX4, which is a CXCR4 mutant with the entire N-terminus (codons 2 to 25) deleted, all the TM and extracellular loop (ECL) mutants contain a single amino acid substitution, mostly of alanine, in their respective sites. The panel of mutations at residues near or within the TM helices were chosen based on the following considerations: (i) charged residues such as D97, D171, and E288 may interact with the oppositely charged residues of natural ligands or SMM chemokines; (ii) highly conserved residues among chemokine receptors or analogous to corresponding sites in other GPCRs, such as H79, Y121, W161, Y219, and N298, are known to be functionally important for other GPCRs (16
); and (iii) residues such as P163 may affect the helical conformations of CXCR4. In addition to these TM mutants, several mutants of the ECL residues were used to investigate the role of the ECL in ligand binding. All of the mutants used for the current binding site mapping experiments were previously tested by our laboratory for their activities in SDF-1α binding and signaling and HIV-1 coreceptor activity (18
). Except for the N terminus of CXCR4, which is well documented in the literature for its roles in receptor physiology and pathology (5
), other regions of CXCR4 such as most of the mutated regions focused on the present study are still poorly understood.
Cell surface expression of CXCR4.
We first investigated the question whether a difference in the binding activity of a ligand toward a wild-type versus a mutant receptor could be due to a change in the level of cell surface expression of the receptor. A particular mutant exhibiting a marked reduction in the binding activity could be caused by its poor expression compared with that of wild-type CXCR4. Flow cytometry experiments were performed on all the stably transfected CXCR4 mutants. We found that all the mutants displayed stable expression levels comparable to or higher than those of wild-type CXCR4 (Fig. ).
FIG. 1. Cell surface expression of wild-type and mutant CXCR4. Stably transfected 293 cells with wild-type or mutant CXCR4 were analyzed for cell surface expression of CXCR4 by flow cytometry using anti-CXCR4 MAb 12G5. Bars represent the mean fluorescence intensity (more ...) d-Amino acid-containing SMM chemokines differ from SDF-1α on CXCR4 binding sites.
According to the results for the vMIP-II-based d
-amino-acid-containing SMM chemokine, the binding activity of d
(1-10)-vMIP-II with its N-terminal (1-10) residues replaced with d
-amino acids was reduced by the point mutation of Tyr45
, or Phe292
as well as by the deletion of the N terminus (Table ). While D171A, E288D, and DNX4 decreased the percent specific binding of d
(1-10)-vMIP-II by 32 to 41%, F87A reduced d
(1-10)-vMIP-II binding by more than 60% (Fig. ). Y45A, D97A, Y121A, W252A, Y255A, and F292A all reduced the binding activity of d
(1-10)-vMIP-II by 13 to 26%. In contrast, SDF-1α did not require Tyr45
, and Glu288
for its interaction with CXCR4, as their mutations had little effect on SDF-1α binding. However, F87A, D171A, and F292A impaired the binding affinity of SDF-1α by 24 to 31%, whereas DNX4 decreased SDF-1α binding by 53% (Fig. ). Note that the current results on the binding activity of SDF-1α obtained using the anti-CXCR4 MAb 12G5 are identical to those of 125
I-labeled SDF-1α binding experiments (18
), demonstrating that the inhibition of 12G5 can substitute for the inhibition of SDF-1α at least for the CXCR4 mutants examined in this study. In fact, according to 125
I-SDF-1α competition binding assays (18
), all of the CXCR4 mutants had no effect on SDF-1α binding, as they showed comparable 50% inhibitory concentrations (~3 nM) to that of wild type. Only three TM mutants, F87A, D171A, and F292A, drastically reduced the binding activity of SDF-1α, as their binding curves did not reach a plateau of nonspecific binding even at 300 nM of unlabeled SDF-1α. Furthermore, the finding of 50% inhibitory concentrations of each CXCR4 mutant comparable to that of the wild type undermines any notion that some mutations, such as Trp161
, may be able to increase the binding activity of SDF-1α.
Residues involved in the chemokine binding and HIV-1 coreceptor activities of CXCR4
FIG. 2. Specific binding of d(1-10)-vMIP-II (A) and SDF-1α (B) to wild-type CXCR4 and mutants. (A) The point mutation of Tyr45, Phe87, Asp97, Tyr121, Asp171, Trp252, Tyr255, Glu288, or Phe292 reduced d(1-10)-vMIP-II binding. The binding activity of d (more ...)
Similar to d(1-10)-vMIP-II, the introduction of d-amino acids in SDF-1α caused the new analogs to interact with a different set of residues on CXCR4 (Table ). For instance, compared with SDF-1α, d(1-8)-SDF-1α required three new residues, namely Tyr45, Asp187, and Glu288, as their mutations reduced d(1-8)-SDF-1α binding activity by 13 to 29% (Fig. ). Also the fact that Phe292, an important residue in SDF-1α binding, was no longer required for d(1-8)-SDF-1α binding further illustrates the differences in the CXCR4 binding sites of SDF-1α versus d(1-8)-SDF-1α. Furthermore, D171A and DNX4 showed a significant difference in their effects on the binding affinities of SDF-1α and d(1-8)-SDF-1α. Whereas D171A reduced SDF-1α binding by 53%, it decreased the percent specific binding of d(1-8)-SDF-1α by a smaller margin, 27%. In contrast, although DNX4 impaired SDF-1α binding by 27%, it caused a greater reduction, 43%, in d(1-8)-SDF-1α binding.
FIG. 3. Binding activities of d(1-8)-SDF-1α (A) and d(1-10)-vMIP-II-(9-68)-SDF-1α (B) to wild-type CXCR4 and mutants. (A) The binding activity of d(1-8)-SDF-1α was reduced by Y45A, F87A, D171A, D187A, E288D, and DNX4. (B) The point mutation (more ...)
In addition, by replacing the N-terminal (1-8) sequence module of SDF-1α with all d forms of (1-10) residues of vMIP-II, the binding site of the new analog could be deviated further away from that of SDF-1α. Besides the overlapping residues also required for SDF-1α binding, d(1-10)-vMIP-II-(9-68)-SDF-1α needed several additional residues, including Tyr45, Asp97, Tyr121, Asp187, Tyr219, Trp252, Tyr255, Asp262, and Glu288 (Table ). In fact, d(1-10)-vMIP-II-(9-68)-SDF-1α was by far the most selective inhibitor, which seems to be consistent with the largest number of distinct residues observed in our binding site mapping experiments. For example, Y45A, F87A, Y121A, E288D, F292A, and DNX4 impaired d(1-10)-vMIP-II-(9-68)-SDF-1α binding by 40 to 70% (Fig. ). The other mutants, including D97A, D171A, D187A, Y219A, W252A, Y255A, and D262A, reduced the binding activity of d(1-10)-vMIP-II-(9-68)-SDF-1α by 19 to 38%. In particular, F87A, F292A, and DNX4, which were also implicated in SDF-1α binding, had a greater impact on the binding activity of d(1-10)-vMIP-II-(9-68)-SDF-1α, as they decreased the percent specific binding of d(1-10)-vMIP-II-(9-68)-SDF-1α by 70, 56, and 64%, respectively. The same set of mutants impaired SDF-1α binding by smaller margins of 24, 31, and 53%, respectively.
d-Amino acid-containing SMM chemokines differ from their l counterparts on CXCR4 binding sites.
d(1-10)-vMIP-II and d(1-10)-vMIP-II-(9-68)-SDF-1α also showed major differences in their binding sites from their l counterparts, namely vMIP-II and (1-10)-vMIP-II-(9-68)-SDF-1α. The main sites involved in the binding activities of vMIP-II and (1-10)-vMIP-II-(9-68)-SDF-1α consist of Phe87, Asp97, Tyr121, Phe292, and the N terminus (Table ). F87A, D97A, Y121A, and F292A reduced vMIP-II binding by 13 to 32%, whereas DNX4 decreased the percent specific binding of vMIP-II by more than 50% (Fig. ). In the case of (1-10)-vMIP-II-(9-68)-SDF-1α, all of the mutants had very little effect on its binding, with the exception of DNX4, which decreased (1-10)-vMIP-II-(9-68)-SDF-1α binding by 52% (Fig. ). In contrast, there were several distinct TM residues necessary for the binding activities of d(1-10)-vMIP-II and d(1-10)-vMIP-II-(9-68)-SDF-1α only (Table ). For instance, compared with its l counterpart, d(1-10)-vMIP-II-(9-68)-SDF-1α binding required a large number of distinct residues such as Tyr45, Phe87, Asp97, Tyr121, Asp171, Asp187, Tyr219, Trp252, Tyr255, Asp262, Glu288, and Phe292. Likewise, five additional TM residues, including Tyr45, Asp171, Trp252, Tyr255, and Glu288, were involved in d(1-10)-vMIP-II binding, unlike in vMIP-II binding. Note that d(1-10)-vMIP-II-(9-68)-SDF-1α and d(1-10)-vMIP-II share a great overlap in their binding sites likely due to a common major binding determinant, the d-amino acid sequence derived from the same N terminus of vMIP-II in these two molecules.
Comparison of CXCR4 binding sites of d-amino-acid-containing SMM chemokines (d-ligands) versus SDF-1α and other l-amino-acid-containing chemokines (l-ligands)
FIG. 4. Inhibition of anti-CXCR4 12G5 binding by vMIP-II (A) and (1-10)-vMIP-II-(9-68)-SDF-1α (B) to wild-type CXCR4 and mutants. The data represent the mean values of three independent assays with the error bars indicating the standard deviations. (A) (more ...) d-Amino-acid-containing SMM chemokines significantly overlap with HIV-1 gp120 on CXCR4 binding sites.
Based on our previous CXCR4 mutational study (18
, and the N terminus are all known to play key roles in the HIV-1 coreceptor activity of CXCR4 (Table ). We notice that Asp187
is the only ECL residue involved not only in d
-ligand binding but also in HIV-1 coreceptor activity, suggesting that it will be important to examine further whether the ECL2, a major loop involved in HIV-1 entry but not in SDF-1α binding (19
), is an important binding region for d
-ligands. We also note that Glu288
is required for the binding activities of all of the d
-ligands, which makes sense considering that Glu288
is located close to the surface of the TM “barrel” (Fig. ). In addition, the other distinct sites required for both HIV-1 gp120 and d
-ligands, such as Tyr45
, and Tyr255
, are located on the upper part of the TM barrel close to the extracellular side or to the ECL2. Their role is likely to be involved with direct interactions with different ligands. Based on the present findings, one can hypothesize that certain flexible determinants of HIV-1 gp120, which can reach into CXCR4 TM domains, may be blocked by d
-ligands that directly interact with these TM residues. Alternatively, it is possible that the TM mutations may cause changes in the conformations of CXCR4 core domains and thus indirectly affect CXCR4 interactions on its surface with HIV-1 gp120 or d
-amino-acid-containing SMM chemokines. In such a case, the potential conformational changes caused by the mutations seem to be selective in hindering CXCR4 interactions with HIV-1 gp120 or d
-ligand interaction, since SDF-1α binding to the mutant receptors was not affected. If this notion of conformational changes were true, this would strongly suggest that different conformations of CXCR4 are functionally important for d
-amino-acid-containing SMM chemokine and SDF-1α. Despite this preferential overlapping in the CXCR4 binding residues of SMM chemokines with HIV-1 over SDF-1α, we note that several mutants of CXCR4, including H79A, P163A, F189A, P191A, E268A, Q272A, H294A, and N298A, significantly reduced the coreceptor activity of CXCR4 (18
) without reducing the binding activities of any ligands (including d
-amino-acid-containing SMM chemokines and SDF-1α), indicating that the interaction of CXCR4 with HIV-1 gp120 involves an extensive set of residues, many of which are not required for the interaction with SMM chemokines or SDF-1α. Nevertheless, the findings from the present study provide a basis for the development of new inhibitory agents, as the CXCR4 binding sites shared by both HIV-1 gp120 and d
-amino-acid-containing ligands may serve as a major target for the development of new HIV-1 inhibitory agents that can reduce or avoid the side effects in binding to the CXCR4 sites important for its normal ligand, SDF-1α.
Comparison of CXCR4 binding sites of SMM chemokines including d-ligands versus HIV-1 gp120 and SDF-1α
FIG. 5. Distinct functional sites for SDF-1α and SMM chemokines highlighted on a hypothetical structural model of CXCR4. As detailed in Table , the residues involved in both SDF-1α and SMM chemokine binding are highlighted in the (more ...) Implications for the design of new selective HIV-1 inhibitors.
We reported here that SMM chemokines (particularly those unnatural d-amino-acid-containing analogs) share many CXCR4 binding sites with HIV-1 gp120 and yet differ from SDF-1α. These results suggest that these chemically engineered molecules have interesting and unique receptor binding mechanisms distinct from those of the natural chemokines and may be used to selectively disrupt the coreceptor activity of CXCR4. This notion is supported by the finding that some of these d-amino-acid-containing SMM chemokines show greater efficacy than SDF-1α in inhibiting HIV-1 entry via CXCR4 (unpublished data). The distinct residues required for the binding activities of d-amino-acid-containing SMM chemokines include Tyr45, Asp97, Tyr121, Asp187, Tyr219, Trp252, Tyr255, Asp262, and Glu288, many of which play important roles in HIV-1 coreceptor activity. These overlapping functional sites for HIV-1 gp120 and d-amino-acid-containing SMM chemokines, located on CXCR4 TM and extracellular domains, may be used to guide the effort to design selective HIV-1 inhibitors that do not interfere with the normal SDF-1α function.