Structural basis for highly effective HIV-1 neutralization by CD4-mimetic miniproteins revealed by 1.5 Å cocrystal structure of gp120 and M48U1

Abstract

The interface between the HIV-1 gp120 envelope glycoprotein and the CD4 receptor contains an unusual interfacial cavity, the "Phe43 cavity", which CD4-mimetic miniproteins with nonnatural extensions can potentially utilize to enhance their neutralization of HIV-1. Here, we report cocrystal structures of HIV-1 gp120 with miniproteins M48U1 and M48U7, which insert cyclohexylmethoxy and 5-hydroxypentylmethoxy extensions, respectively, into the Phe43 cavity. Both inserts displayed flexibility and hydrophobic interactions, but the M48U1 insert showed better shape complementarity with the Phe43 cavity than the M48U7 insert. Subtle alteration in the gp120 conformation played a substantial role in optimizing fit. With M48U1, these translated into a YU2-gp120 affinity of 0.015 nM and neutralization of all 180 circulating HIV-1 strains tested, except clade-A/E isolates with noncanonical Phe43 cavities. Ligand chemistry, shape complementarity, surface burial, and gp120 conformation act in concert to modulate binding of ligands to the gp120-Phe43 cavity and, when optimized, can effect near-pan-neutralization of HIV-1.

Figure 1. Surface-plasmon resonance (SPR) analysis of the binding of CD4-mimetic miniproteins, M48U1 and M48U7, to HIV-1 YU2 gp120

A very slow dissociation rate contributes to the extraordinary affinity of M48U1 for gp120. M48U7 binds with a similar on-rate but dissociates from gp120 about 10-times faster than M48U1. SPR profiles of the binding of (A) M48U1 and (B) M48U7 to full-length HIV-1 gp120 from the clade B YU2 strain. The black lines indicate independent injections of the CD4-mimetic miniproteins with concentrations sampled at 2-fold dilution. Concentrations from 3 nM to 0.006 nM and from 8 nM to 0.06 nM were sampled for M48U1 and M48U7, respectively. Each concentration was sampled in duplicate. The red lines show the global fit of the data to a Langmuir 1:1 binding model. SPR profile of HIV-1 YU2 gp120 binding to M48U1 immobilized on a CM5 chip is shown in Figure S1.

Figure 2. Structures of M48U1 and M48U7 bound to HIV-1 gp120 show ligand inserts penetrating and filling the gp120 Phe43 cavity

(A) M48U1 (red and green) binds to a conserved gp120 cavity at the intersection of the outer domain (wheat), inner domain (slate) and bridging sheet (pink). (B) M48U7 (red and cyan) binds to the same site on gp120 as M48U1. gp120 is shown as grey surface with the CD4 footprint on gp120 colored yellow. (C) Zoomed-in view of the M48U1 binding site showing hydrogen bonding (black dotted lines) of M48U1 Arg 9 with the conserved Asp 368 of gp120. Figures (A) and (C) are related by an approximately 90° rotation about a horizontal axis. (D) gp120 cross-section showing the U1 side chain (green sticks with the oxygen atom colored red) penetrating the Phe43 cavity. Figures (A) and (D) are related by an approximately 90° rotation about a vertical axis. (E) Zoomed-in view of the M48U7 binding site showing the salt bridge (black dotted) of M48U7 Arg 9 with gp120 Asp 368. Figures (B) and (E) are related by an approximately 90° rotation about a horizontal axis. (F) gp120 cross-section showing U7 side chain (cyan sticks with the oxygen atom colored red) penetrating the Phe43 cavity. The blue mesh surrounding is the 2Fo-Fc map contoured at 2 sigma for M48U1 and 1.6 sigma for M48U7. Figures (B) and (F) are related by an approximately 90° rotation about a horizontal axis.

Figure 3. Interactions of M48U1 and M48U7 with the gp120 Phe43 cavity

Flexible inserts use shape and chemical complementarity to optimize fit to the Phe43 cavity. (A) M48U1 (red and green) binds to a conserved gp120 cavity at the intersection of the outer domain (wheat), inner domain (slate) and bridging sheet (pink). Red spheres are water molecules. (B) M48U7 (red and cyan) binds to the same site on gp120 as M48U1. gp120 is shown as grey surface. The black dotted lines show hydrogen bonds made by the terminal hydroxyl group of residue 23_M48U7 side-chain in the Phe43 cavity. (C) and (D) are 90° rotated views of (A) and (B), respectively. (E) Zoomed-in view of U1 moiety inserting into the Phe43 cavity. (F) Zoomed-in view of U7 moiety inserting into the Phe43 cavity. CD4 binding footprint is colored yellow. In the multilayer transparent surface representation shown in (E) and (F), the outer surface is gp120 and the inner surface is the ligand. Interactions of M48U1 and M48U7 outside the Phe43 cavity are shown in Table S2.

Figure 4. Distance-sorted difference distance matrices reveal conformational changes in the gp120 Phe43 cavity as a result of M48U1 and M48U7 binding

gp120 Phe43 cavity bound to M48U1 or M48U7 shows greater resemblance to the Phe43 cavity in unliganded gp120 than the Phe43 cavity in gp120 bound to CD4 and CD4-induced monoclonal antibody 17b. Distance-sorted difference distance matrices of gp120 were constructed using coordinates of atoms lining the gp120 Phe 43 cavity selected with fpocket. Atoms within 6 Å of U1 were sorted in increasing order of distance from the Phe43 Cα atom of CD4 and are shown along with the corresponding gp120 residue in the far-left panel. The columns in this panel, from left to right, denote distances in Å from Cα of CD4 Phe43, residue names, residue numbers, and atom names. Difference distance matrices composed of these gp120 residues were calculated for unliganded YU2 gp120 and for various gp120 complexes with CD4, M48U1, and M48U7. Each i,j matrix element shows the distance between atom i and atom j in the first specified structure minus the distance between the same atoms in the second specified structure. The difference distance matrices was quantified by mean difference distances, and is shown for each comparison. Physical and chemical properties of the gp120 Phe43 cavity properties are summarized in Figure S3.

Figure 5. Ligand induced motion in the gp120 Phe43 cavity

A cross-section of gp120 is shown as a transparent grey surface. Phe43 cavity atoms are shown as spheres, and the residues connecting them as sticks. Distance-sorted difference distance matrices of gp120 Phe43 cavity, bound to different ligands shown in Figure 4, were used to calculate net motion at each cavity atom. The cavity atoms were colored with a yellow-white-magenta gradient, where yellow represents least motion and magenta represents highest differences between (A) gp120 bound to CD4 (yellow) and 17b IgG vs unliganded gp120, (B) gp120 bound to M48U1 vs unliganded gp120, (C) gp120 bound to M48U1vs gp120 bound to CD4 and 17b IgG, (D) gp120 bound to M48U7 (red and cyan) vs unliganded gp120, and (E) gp120 bound to M48U1vs gp120 bound to CD4 and 17b IgG. Analysis of Phe43 cavity volume shown in Figure S4.

Figure 6. Comparison of Phe43 cavity conformation between different CD4-mimetic miniproteins

Distance-sorted difference distance matrices of gp120 were constructed using the same set of atoms as in Figure 4. Difference distance matrices were calculated to compare Phe43 cavity conformations of gp120 bound to CD4-mimetic miniproteins M48, M47, M48U1 or M48U7, relative to each other. Each i,j matrix element shows the distance between atom i and atom j in the first specified structure minus the distance between the same atoms in the second specified structure. The difference distance matrices was quantified by overall mean of the difference distances, and is shown for each comparison. Structural changes in the Phe43 cavity conformation upon binding to different cavity filling ligands is shown in Figure S5.

Figure 7. HIV-1 neutralization by CD4-mimetic peptides M48U1 and M48U7

Neutralization profiles of (A) M48U1 and M48U7 depicted with 24-isolate dendrograms and (B) M48U1 depicted with 180-isolate dendrogram representative of circulating HIV-1 Tier 1 and Tier 2 viruses. Neutralization IC₅₀ values are tabulated in Table S3. Neutralization dendrograms of sCD4, M48 and M47 are shown in Figure S5. The molecular weights of M48U1 and M48U7 are 3048 Da and 3038 Da, respectively.