Structure of the Carboxyl-Terminal Dimerization Domain of the HIV-1 Capsid Protein

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Science  31 Oct 1997:
Vol. 278, Issue 5339, pp. 849-853
DOI: 10.1126/science.278.5339.849


The carboxyl-terminal domain, residues 146 to 231, of the human immunodeficiency virus–1 (HIV-1) capsid protein [CA(146–231)] is required for capsid dimerization and viral assembly. This domain contains a stretch of 20 residues, called the major homology region (MHR), which is conserved across retroviruses and is essential for viral assembly, maturation, and infectivity. The crystal structures of CA(146–231) and CA(151–231) reveal that the globular domain is composed of four helices and an extended amino-terminal strand. CA(146–231) dimerizes through parallel packing of helix 2 across a dyad. The MHR is distinct from the dimer interface and instead forms an intricate hydrogen-bonding network that interconnects strand 1 and helices 1 and 2. Alignment of the CA(146–231) dimer with the crystal structure of the capsid amino-terminal domain provides a model for the intact protein and extends models for assembly of the central conical core of HIV-1.

The 26-kD capsid protein (CA) performs essential roles both early and late in the life cycle of HIV. Capsid is initially translated as the central region of the 55-kD Gag polyprotein, where it functions in viral assembly (1, 2) and in packaging the cellular protein cyclophilin A (3). As the virus buds, Gag is processed by the viral protease to produce three discrete new proteins—matrix, capsid, and nucleocapsid—as well as several smaller peptides. After capsid has been liberated by proteolytic processing, it rearranges into the conical core structure that surrounds the viral genome at the center of the mature virus (1, 4). Genetic analyses have revealed that capsid also performs essential roles as the virus enters, uncoats, and replicates in the new host cell (2), although these early steps in viral replication are currently poorly understood.

Capsid is composed of two distinct domains (2, 5, 6). The NH2-terminal domain (residues 1 to 145) contributes to viral core formation and binds cyclophilin A. In contrast, the COOH-terminal domain is required for capsid dimerization (5,6), Gag oligomerization (7), and virion formation (2). This domain also contains a conserved stretch of 20 amino acids, termed the MHR, that is found in all known onco- and lentiviruses and in the yeast retrotransposon Ty-3 (8, 9) (Fig. 1). As would be expected from this conservation, the MHR is essential for viral replication, although its precise functions remain unclear because different MHR mutations block viral replication at distinct stages, including assembly (9-12), maturation (10, 12), and early steps of infectivity (9, 11, 12).

Figure 1

HIV-1 CA(151–231) secondary structure and sequence alignment with representative onco- and lentiviruses. Disordered residues are indicated with a dashed line. The MHR is indicated with a bar, and invariant residues in this alignment are denoted with asterisks. Secondary structures are color-coded as follows: red, helix 1 (residues 11 to 24); magenta, helix 2 (29 to 42); cyan, helix 3 (46 to 54); green, helix 4 (61 to 67). SIV, simian immunodeficiency virus; RSV, Rous sarcoma virus; HTLV-I, human T cell leukemia virus–type I; MMTV, mouse mammary tumor virus; MPMV, Mason-Pfizer monkey virus; MMLV, Moloney murine leukemia virus. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

In an effort to understand the structural basis for capsid dimerization and MHR conservation, we determined the crystal structure of selenomethionine-substituted CA(151–231) at 1.7 Å resolution (13, 14) and native CA(146–231) at 3.0 Å resolution (15) (Tables 1 and 2). These two structures are similar, and therefore only CA(151–231) is described in detail, except where the capsid dimer interface is discussed (see below). The CA(151–231) model includes the initiator Met151 residue (which replaces Leu151 in the native sequence), capsid residues 152 to 220, and 63 ordered water molecules. The last 11 residues of capsid were not visible in electron density maps and have been omitted from the model. The Rvalue is 22.9% and the R free value is 27.4%.

Table 1

Diffraction data. Parentheses denote highest shells of resolution.

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Table 2

Refinement statistics for CA(151–231). The model has 70 amino acid residues and 63 water molecules.

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Each CA(151–231) molecule is composed of an extended strand followed by four α helices (Fig. 2A) (16). The domain adopts an ovoid fold with overall dimensions of 27 Å by 29 Å by 38 Å. The MHR forms a compact strand-turn-helix motif that packs against the COOH-terminal end of helix 2 (Fig. 2B). The four most highly conserved MHR residues (Gln155, Gly156, Glu159, and Arg167) form a hydrogen-bonding network that stabilizes this structure and links it to helix 2. Within the network, the Gln155 side chain bridges the NH2-terminus of helix 1 and the COOH-terminus of helix 2. The side chains of Glu159 and Arg167 form hydrogen bonds to main-chain amide and carbonyl groups across the MHR loop and also form a buttressing salt bridge. Finally, both main chain heteroatoms of Gly156 also form a hydrogen bond across the loop. The conservation of Gly156 can be explained because larger side chains would clash sterically with the Glu159 Cβ atom, particularly as the conserved Pro157 restricts the local chain flexibility. The structure also explains the observation that conservative mutations of invariant capsid MHR residues (for example, Gln155 → Asn or Glu159 → Asp) block HIV-1 replication (10), because even these single carbon mutations are expected to disrupt the hydrogen-bonding network that defines the MHR fold.

Figure 2

(A) Structure of the CA(151–231) monomer. Color code is the same as in Fig. 1, with the disulfide bond between Cys198 and Cys218 shown in yellow. Residues 156 to 158 form a type I β-turn and residues 206 to 209 form a type II β-turn. (B) Expanded view of the MHR. The first and last residues of the MHR (residues 153 and 172) are indicated by arrows. The hydrogen-bonding interactions of the four most conserved MHR residues (Gln155, Gly156, Glu159, and Arg167) are shown explicitly. The following apparent hydrogen-bonding interactions (donor-acceptor distance of <3.1Å) are illustrated: Glu159N···Gly156 O, Gln155Nɛ2···Glu159 O, Asn195N···Gln155 Oɛ1, Gly156N···Glu159 Oɛ1, Arg167Nη1···Glu159 Oɛ1, Arg167Nη1···Arg154 O, and Arg167Nɛ···Glu159 Oɛ2 (Figs. 2, 4, and 5 were made with MOLSCRIPT and RASTER 3D).

A series of conserved hydrophobic residues (Phe161, Tyr164, Val165, Tyr169, and Leu172) are also essential for MHR function (8,9). As predicted (17), these residues lie on one face of helix 1, where they contribute to the hydrophobic core of the protein. Thus, all of the conserved MHR residues perform critical structural roles. The remarkable conservation of the 20-residue MHR within the highly variable Gag polyprotein suggests that the MHR structure mediates an essential interaction with a highly conserved binding partner, such as a cellular factor or an invariant segment of a viral protein.

Dimerization of the intact capsid protein and the two COOH-terminal domain fragments was quantitated by equilibrium sedimentation. Native capsid exhibited a monomer-dimer equilibrium that was best fit with a dissociation constant K d = 18 ± 1 μM (Fig. 3A), in good agreement with previous studies (6, 18). The CA(146–231) protein dimerized with nearly the same affinity as the full-length protein (K d = 10 ± 3 μM; Fig. 3B). Surprisingly, however, the slightly shorter CA(151–231) protein did not dimerize significantly, even at concentrations up to 100 μM (19). We therefore conclude that CA residues 146 to 151 are necessary for forming the high-affinity capsid dimer interface.

Figure 3

Representative sedimentation profiles of (A) intact CA, (B) CA(146–231), and (C) CA(M185A) mutant protein. The analysis reveals that intact CA and CA(146–231) dimerize with similar affinities (K d = 18 ± 1 and 10 ± 3 μM, respectively), but that the CA(M185A) mutation abolishes dimerization. Theoretical curves for monomer (M) and monomer/dimer (M/D) distributions are shown assuming K d = 18 μM (A and C) or 10 μM (B). A 280, absorbance at 280 nm.

Both CA(146–231) and CA(151–231) formed dimers in the crystal through the packing of helix 2 across crystallographic twofold axes. The two dimers differ, however, by an apparent ∼30° rotation of individual monomers (Fig. 4). The different dimerization geometries result from additional intermolecular contacts made by residues 146 to 151 (15). Our equilibrium sedimentation experiments indicate that the CA(146–231) dimer represents the structure of the high-affinity dimer interface of the intact protein (Fig. 3). This interface is created by the parallel packing of helix 2 against its symmetry-related mate, creating a hydrophobic core that includes residues Val181, Trp184, Met185, and Leu189. The interface does not use MHR residues, which indicates that the MHR and dimerization motif are distinct entities that likely perform different functions.

Figure 4

(A) Structure of the CA(151–231) dimer. View is along the dyad axis, that is, from the top of Fig. 2A. (B) CA(146–231) dimer with the side chains of Trp184 and Met185 shown explicitly. Color code is the same as in Figs. 1 and 2.

We used site-directed mutagenesis to confirm that the dimer interface of the COOH-terminal CA(146–231) domain is also the principal dimer interface of the intact HIV-1 CA. Residue Met185, which is in the center of the CA(146–231) dimer interface, was mutated to Ala in the context of the intact CA. This mutant protein (M185A) was monomeric at all concentrations tested (Fig.3C) (20). A second dimer interface mutation, Trp184 → Ala (W184A), similarly abolished capsid dimerization. Thus, helix 2 forms the principal dimer interface of the intact capsid protein.

We also tested the effect of the capsid M185A mutation on viral replication. This mutation reduced, but did not eliminate, the production of HIV-1(NL4–3) viral particles from 293T human embryonic kidney cells (21). The viral particles that were produced, however, were completely noninfectious. Thus, a point mutation that eliminates capsid dimerization in vitro also blocks viral replication in culture, which indicates that the crystallographically defined capsid dimer interface plays an essential role in HIV-1 replication.

The structures of both domains of HIV-1 CA are now known (5, 22,23), and we have therefore examined possible models for the structure of the intact protein. The final ordered residue in both the crystal and nuclear magnetic resonance structures of capsid residues 1 to 151 [CA(1–151)] was Tyr145 (5, 23). Thus, we modeled the intact CA by connecting the known CA(1–145) and CA(146–231) domains through a five-residue linker sequence corresponding to residues 146 to 150 (Fig.5).

Figure 5

Model for the intact HIV-1 capsid dimer. The CA(146–231) dimer (cyan) is shown covalently linked to the CA(1–151) domain (23). The five disordered residues that connect the two domains can be modeled to allow ∼90° range of relative rotational orientation for the two domains about the vertical twofold axis.

In the crystal structure of CA(1–151) in complex with cyclophilin A, the CA molecules associate into continuous planar strips that exhibit two distinct twofold symmetric interfaces (23). We speculate that the more extensive of these two NH2-terminal domain interfaces (the β-hairpin interface) may cooperate with the COOH-terminal dimer interface described here to mediate higher order capsid protein assembly. Repetition of these two capsid dimer interfaces would create a strip of capsid molecules that could then wind up to create the core of HIV.

  • * These authors contributed equally to this report.


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