3.1. Structural Basis for the Lack of gp120-Binding by Murine CD4
Previous experiments have revealed that the mCD4 protein does not interact with HIV-1 gp120 [16
]. To identify the structural properties responsible for this lack of interaction, a homology model of the mCD4-gp120 complex was generated.
Homology modeling of mCD4 is hampered by the fact that slightly different sequence alignments between hCD4 and mCD4 have been reported in the past [39
], which differ in the alignment of the C″
D-loop (Figure S1). To identify the alignment, which is more suitable for comparative modeling, two structures were generated based on the different sequence alignments and compared with respect to their model quality (Table S1, Figures S2, and S3). Interestingly, both models exhibit clashes at the mCD4-gp120 interface, which can, therefore, be considered as an intrinsic feature impeding interaction of the complex regardless of the CD4 sequence alignment used. Therefore, all further analysis is based on the murine model 1, which exhibits the more favorable structural properties (Table S1).
The overlay between the murine and human CD4-gp120 complexes reveals the high overall structural similarity (Figure ), while a detailed inspection of the interface highlights several clashes present in the mCD4-gp120 complex (Figure ). Clashing residues are predominantly located in the loops flanking β
-D-loop), two regions that show sequence insertions in mouse compared to human CD4 (). Structural analysis identifies several residues that form clashes >0.8
Å (). The largest intermolecular clash is found between G49, located in the C″
D-loop, and S365, located in the CD4-binding loop of gp120, with a size of 1.95
Å. An attempt to remove this clash by a 500-ps MD simulation in explicit solvent resulted in a very unfavorable backbone geometry of G49:
= +159.0° and ψ
= −86.6° (compared to
= −81.2°, ψ
= +27.2° in the initial model). Furthermore, K42 and V44 contribute significant clashes to the interface. K42, an insertion in the C′C″
-loop, clashes with two residues of the bridging sheet of gp120. The larger of the clashes is formed between the K42 (mCD4) and K429 (gp120) side chains (, ) indicating that electrostatic repulsion additionally counteracts the mCD4-gp120 complex formation.
Figure 2 Clashes of murine CD4 with gp120. (a) Overlay of the unbound mCD4 model onto the hCD4-gp120 complex structure 1RZJ . (b) Close-up of the clash area. Clashes are predominantly found for residues in the loops flanking β-strand C″ (G48, (more ...)
Table 1 Clashes of mCD4 with gp120. WHAT_CHECK analysis of clashes detected between mCD4 with gp120. All clashes > 0.8Å are listed; bb and sc denote clashes by backbone or side-chain atoms. The clashes are visualized in .
Valine44, the sequential equivalent to F43 in hCD4, overlaps with I371. Interestingly, the described clashes affect residues and positions where human and mouse sequences diverge. Those findings are in line with previous studies that demonstrated a complete loss of binding affinity following K- and GS-insertion in C″
-flanking loops of hCD4 [39
]. In conclusion, clashes of the loops flanking strand C″
, that are longer in mCD4, are most likely the major reason for impeded gp120-binding of mCD4.
The fact that gp120 mainly forms clashes with the longer loops of mCD4 prompted us to investigate whether these clashes might be removed in an mCD4 mimetic peptide (mCD4-M) due to its larger conformational freedom. Interestingly, energy minimization is sufficient to remove all intermolecular clashes (>0.4
Å) between such a peptide and gp120. In order to ensure that this finding is not an artifact of the applied force field, the respective peptide was synthesized and its gp120-binding affinity was verified experimentally.
3.2. Experimental Investigation of the gp120-Binding of CD4-Derived Peptides
Peptides comprising residues 22–66 of mCD4 (termed mCD4-M) and 22–64 of hCD4 (termed hCD4-M), respectively, were synthesized and their gp120-binding affinities experimentally determined (; ). This experiment shows that mCD4-M is indeed capable of binding to gp120, and that this interaction is only slightly weaker than that of the homologous hCD4-M peptide.
Figure 3 Binding affinities of CD4 peptides. Relative affinities (hCD4-M = 1) to HIV-1 gp120 of peptides mimicking the putative gp120-binding site of human (hCD4-M) and murine CD4 (mCD4-M), as well as peptide variants in which the hot spot residues were replaced (more ...)
To verify that the binding of the peptides is specific, variants (hCD4-M*, mCD4-M*; ) were generated that address the hot spot residues crucial for gp120-binding known from the gp120-CD4 crystal structures [3
]. In hCD4-M, F43 and R59 were replaced by alanine. Due to sequence diversity, the corresponding residues in mCD4-M are V44 and K61 (). In these peptides, also R58 (K60 in the murine peptide) was replaced by alanine to avoid functional compensation of R59 (K61 in the murine peptide) by the sequentially adjacent basic residue, which might occur in peptides due to a higher flexibility compared to proteins. Binding of either triple variant to gp120 was notably reduced, indicating that mCD4-M specifically targets the CD4-binding site of gp120 (). This suggests that the lack of interaction detected for mCD4 protein is not due to the sequence of mCD4 itself, but most likely to the rigid conformation imposed by the immunoglobulin fold. Therefore, the affinity can at least partially be restored in the more flexible mCD4 peptides.
3.3. Structural Analysis of the mCD4-M-gp120 Interaction
To understand the binding of mCD4-M in greater detail, a 100-ns molecular dynamics simulation was performed for the peptide-gp120 complex. Subsequently, the different key interaction regions were analyzed in greater detail.
The peptide core contains three β
-strands, C, C′, and C″
, assembled in an antiparallel fashion. Stand C″
directly interacts with strand β
15 of gp120 (, Figure ). Hence, those interactions were analyzed over the course of simulation. For the intramolecular C-C′-C″β
-sheet, hydrogen bond distances and torsion angles were monitored over time and are plotted in Figures and . Distances between main chain nitrogen and oxygen atoms range between 2.5–3.0
Å and are almost preserved during simulation indicating stability of core hydrogen bonding (see d1–d4 in Figure ). Analysis of torsion angles
) and ψ
) revealed an ideal β
-sheet backbone geometry for residues V44 to R47 of strand C″
values ranging at negative and positive values characteristic for a β
-sheet. Taken together, preservation of hydrogen bond distances and β
-sheet backbone angles over time guarantees the stability of the peptide core. This intrinsic stability also is the prerequisite for the correct positioning of the peptide at the gp120-interface. Here, the peptide forms three backbone hydrogen bonds to β
15 of the CD4-binding loop of gp120 during simulation time (see d5–d7 in Figure ). Hydrogen bonds are established between residues L45 and R47 of CD4 and D368 and G366 of gp120. One of the contacts (R47(O)-G366(N)) exhibits larger fluctuations; however, a transient hydrogen bonding can be detected over the entire simulation time (distance d7 in Figure ). The three hydrogen bonds are arranged in an antiparallel β
-sheet fashion, thereby extending the C-C′-C″
-sheet by strand β
15 across the interface. When comparing the peptide interface to the human CD4-gp120 complex of the crystal structure, a striking similarity can be noted both with respect to the β
-sheet structure and the hydrogen bonding pattern (Figure ). The weaker third hydrogen bond detected in the mCD4-M-gp120 interface also exhibits a nonoptimal geometry in the hCD4-gp120 interface thus supporting our results [4
Figure 4 Intrinsic stability and gp120-interaction of the mCD4 peptide core. (a) Scheme of intra- and intermolecular hydrogen bonds and analyzed distances and torsion angles. Measured distances are marked d1–d7. Residues selected for torsion analysis are (more ...)
In addition to the intermolecular β
-sheet structure, the interface is stabilized by tight van der Waals packing of V44 into a cavity of gp120 as shown in . Tight packing is also reflected in the good interaction energy of V44 which is dominated by a strong van der Waals term (). In contrast to the mCD4 protein, where V44 causes interface clashes ( and ), the conformational plasticity of the peptide allows the residue to penetrate into the cavity. This interaction is structurally reminiscent to that of F43 in human CD4, which extends deeply into the F43-cavity of gp120 [4
], forming a key contact of the hCD4-gp120 interface. An F43A mutation in human CD4 leads to a dramatic reduction (500-fold) of gp120-binding [11
Figure 5 Van der Waals packing of V44 at the peptide-gp120 interface. (a) Enlargement of the interface. V44 of β-strand C″ packs tightly into a pocket of the gp120 surface. (b) Replacement by alanine leads to loss of van der Waals packing. (c) (more ...)
This data is also in agreement with our experimental data for the peptides showing that replacement of F43, R58, and R59 (hCD4-M*), as well as V44, K60, and K61 (mCD4-M*) with alanine clearly diminished binding to gp120 (). The methyl side chain of alanine cannot compensate for the missing van der Waals packing of the longer hydrophobic valine side chain, resulting in a hole in the pocket (). In conclusion, the stability of the peptide core allows the formation of an intermolecular β-sheet structure and a tight van der Waals packing at the gp120 interface.
The next analysis focused on the loops flanking strand C″ in mCD4-M. These loops, which are longer in mCD4 (), reside at the gp120 interface and form clashes in the complex of full-length mCD4 (). In order to investigate the conformation of these peptide loops upon gp120-binding, the torsion angles of the respective residues were monitored over simulation time. Contrary to the conformationally stable strand C″, the flanking loops exhibit much greater fluctuation of their backbone geometry over time (). A pronounced flexibility of backbone dihedral angles is detected for residues G41 and K42, located in the C′C″-loop, and residues G48 and G49, residing in the C″D-loop. The higher loop flexibility also offers an explanation for the absence of clashes in mCD4-M, which are observed for the mCD4 protein (). In addition, the conformational flexibility of K42 allows to remove the electrostatic repulsion that is observed between K42 and K429 of gp120 in the complex with the mCD4 protein.
Figure 6 Fluctuations of torsion angles in loops flanking strand C″ of mCD4-M. (a) Schematic presentation of the mCD4-M peptide topology. Analyzed loops are colored red. (b) Dihedral angles of the loops flanking strand C″ as function of simulation (more ...)
An enhanced flexibility is also observed for the carboxy-terminal strand D (Figures and ), which is involved in both intramolecular interactions with strand C and in gp120-binding. In contrast to the flanking loops analyzed above, strand D does not only exhibit enhanced local fluctuations but instead becomes completely detached from the mCD4-M core and the gp120 interface. As a consequence, the intermolecular K61-D368 salt-bridge, which is considered an important element of the gp120-CD4 complex, is lost after 6
ns. Consequently, the intrinsic peptide stability is reduced and the gp120-peptide interface weakened. However, transient contacts detected between residues K60/K61 and several alternative gp120 residues may at least partially compensate for the lost K61-D368 salt-bridge ().
Figure 7 Contacts of K60 and K61 of mCD4-M at the gp120 interface. (a) Interaction energy of K60 and K61 during the simulation. Total energy, electrostatic, and van der Waals contibution are colored in black, blue, and red respectively. (b) Visualization of inter- (more ...)
Figure 8 Optimization of mCD4-M. (a) mCD4-peptide-gp120 complex at the beginning (left) and after 100-ns MD simulation (right). For color-coding refer to . (b) Visualization of engineered disulfide bond C65–C23 yielding a cyclic peptide with enhanced (more ...)
Although mCD4-M displays a rigid core aligning optimally at the gp120-interface, simulation revealed strand D to detach (Figures and ), thereby destabilizing the interaction. Therefore, we proposed that fixation of strand D to the peptide core might not only enhance intrinsic peptide stability but also contribute to the preservation of interface key contacts such as the K61-D368 salt-bridge. Structural analysis suggested a disulfide bridge that fixes the flexible C-terminus of mCD4-M to the peptide core. Due to their spatial proximity in the initial model, a peptide containing a disulfide bridge between cysteine residues at positions 23 and 65 was expected to enhance peptide stability ( and ).
Subsequent binding assays involving this cyclic peptide (mCD4-M**) confirmed that it binds gp120 with a significantly higher affinity than linear mCD4-M (), validating the proposed beneficial effect of the conformational constraint introduced by the disulfide bond.
In conclusion, the presented study provides an excellent example of how a combination of computational and experimental methods can be used to shed light on the structural basis of species selectivity in protein-protein interactions, as well as to design and generate molecules with desired qualities. Furthermore, this strategy is of significant potential benefit for the process of structure-based design of synthetic protein mimics, by proposing ways to avoid spatial hindrance, thus improving structural complementarity, and, consequently, affinity between the molecules involved. CD4-derived peptides, which specifically and with high affinity target the CD4-binding site of gp120, are potential candidates for the development of HIV-1 entry inhibitors. The cyclic murine CD4 mimetic peptide proposed here may, therefore, serve as a starting point for the development of such a drug molecule.