Vladimir N. Malashkevich

Inhibiting HIV-1 Entry: Discovery of D-Peptide Inhibitors that Target the gp41 Coiled-Coil Pocket

The HIV-1 gp41 protein promotes viral entry by mediating the fusion of viral and cellular membranes. A prominent pocket on the surface of a central trimeric coiled coil within gp41 was previously identified as a potential target for drugs that inhibit HIV-1 entry. We designed a peptide, IQN17, which properly presents this pocket. Utilizing IQN17 and mirror-image phage display, we identified cyclic, D-peptide inhibitors of HIV-1 infection that share a sequence motif. A 1.5 A cocrystal structure of IQN17 in complex with a D-peptide, and NMR studies, show that conserved residues of these inhibitors make intimate contact with the gp41 pocket. Our studies validate the pocket per se as a target for drug development. IQN17 and these D-peptide inhibitors are likely to be useful for development and identification of a new class of orally bioavailable anti-HIV drugs.

The Crystal Structure of a Five-Stranded Coiled Coil in COMP: A Prototype Ion Channel?

Oligomerization by the formation of α-helical bundles is common in many proteins. The crystal structure of a parallel pentameric coiled coil, constituting the oligomerization domain in the cartilage oligomeric matrix protein (COMP), was determined at 2.05 angstroms resolution. The same structure probably occurs in two other extracellular matrix proteins, thrombospondins 3 and 4. Complementary hydrophobic interactions and conserved disulfide bridges between the α helices result in a thermostable structure with unusual properties. The long hydrophobic axial pore is filled with water molecules but can also accommodate small apolar groups. An “ion trap” is formed inside the pore by a ring of conserved glutamines, which binds chloride and probably other monatomic anions. The oligomerization domain of COMP has marked similarities with proposed models of the pentameric transmembrane ion channels in phospholamban and the acetylcholine receptor.

Short constrained peptides that inhibit HIV-1 entry.

Peptides corresponding to the C-terminal heptad repeat of HIV-1 gp41 (C-peptides) are potent inhibitors of HIV-1 entry into cells. Their mechanism of inhibition involves binding in a helical conformation to the central coiled coil of HIV-1 gp41 in a dominant–negative manner. Short C-peptides, however, have low binding affinity for gp41 and poor inhibitory activity, which creates an obstacle to the development of small drug-like C-peptides. To improve the inhibitory potency of short C-peptides that target the hydrophobic pocket region of gp41, we use two strategies to stabilize the C-peptide helix: chemical crosslinking and substitution with unnatural helix-favoring amino acids. In this study, the short linear peptide shows no significant inhibitory activity, but a constrained peptide (C14linkmid) inhibits cell–cell fusion at micromolar potency. Structural studies confirm that the constrained peptides bind to the gp41 hydrophobic pocket. Calorimetry reveals that, of the peptides analyzed, the most potent are those that best balance the changes in binding enthalpy and entropy, and surprisingly not those with the highest helical propensity as measured by circular dichroism spectroscopy. Our study reveals the thermodynamic basis of inhibition of an HIV C-peptide, demonstrates the utility of constraining methods for a short antiviral peptide inhibitor, and has implications for the future design of constrained peptides.

Structural insight into Parkinson's disease treatment from drug-inhibited DOPA decarboxylase.

DOPA decarboxylase (DDC) is responsible for the synthesis of the key neurotransmitters dopamine and serotonin via decarboxylation of l-3,4-dihydroxyphenylalanine (l-DOPA) and l-5-hydroxytryptophan, respectively. DDC has been implicated in a number of clinic disorders, including Parkinsons disease and hypertension. Peripheral inhibitors of DDC are currently used to treat these diseases. We present the crystal structures of ligand-free DDC and its complex with the anti-Parkinson drug carbiDOPA. The inhibitor is bound to the enzyme by forming a hydrazone linkage with the cofactor, and its catechol ring is deeply buried in the active site cleft. The structures provide the molecular basis for the development of new inhibitors of DDC with better pharmacological characteristics.

Structure of the Bcr-Abl oncoprotein oligomerization domain.

The Bcr-Abl oncoprotein is responsible for a wide range of human leukemias, including most cases of Philadelphia chromosome-positive chronic myelogenous leukemia. Oligomerization of Bcr-Abl is essential for oncogenicity. We determined the crystal structure of the N-terminal oligomerization domain of Bcr-Abl (residues 1–72 or Bcr1–72) and found a novel mode of oligomer formation. Two N-shaped monomers dimerize by swapping N-terminal helices and by forming an antiparallel coiled coil between C-terminal helices. Two dimers then stack onto each other to form a tetramer. The Bcr1–72 structure provides a basis for the design of inhibitors of Bcr-Abl transforming activity by disrupting Bcr-Abl oligomerization.

Side-chain repacking calculations for predicting structures and stabilities of heterodimeric coiled coils.

An important goal in biology is to predict from sequence data the high-resolution structures of proteins and the interactions that occur between them. In this paper, we describe a computational approach that can make these types of predictions for a series of coiled-coil dimers. Our method comprises a dual strategy that augments extensive conformational sampling with molecular mechanics minimization. To test the performance of the method, we designed six heterodimeric coiled coils with a range of stabilities and solved x-ray crystal structures for three of them. The stabilities and structures predicted by the calculations agree very well with experimental data: the average error in unfolding free energies is <1 kcal/mol, and nonhydrogen atoms in the predicted structures superimpose onto the experimental structures with rms deviations <0.7 Å. We have also tested the method on a series of homodimers derived from vitellogenin-binding protein. The predicted relative stabilities of the homodimers show excellent agreement with previously published experimental measurements. A critical step in our procedure is to use energy minimization to relax side-chain geometries initially selected from a rotamer library. Our results show that computational methods can predict interaction specificities that are in good agreement with experimental data.

All‐trans retinol, vitamin D and other hydrophobic compounds bind in the axial pore of the five‐stranded coiled‐coil domain of cartilage oligomeric matrix protein

The potential storage and delivery function of cartilage oligomeric matrix protein (COMP) for cell signaling molecules was explored by binding hydrophobic compounds to the recombinant five‐stranded coiled‐coil domain of COMP. Complex formation with benzene, cyclohexane, vitamin D3 and elaidic acid was demonstrated through increases in denaturation temperatures of 2–10°C. For all‐trans retinol and all‐trans retinoic acid, an equilibrium dissociation constant KD = 0.6 μM was evaluated by fluorescence titration. Binding of benzene and all‐trans retinol into the hydrophobic axial pore of the COMP coiled‐coil domain was proven by the X‐ray crystal structures of the corresponding complexes at 0.25 and 0.27 nm resolution, respectively. Benzene binds with its plane perpendicular to the pore axis. The binding site is between the two internal rings formed by Leu37 and Thr40 pointing into the pore of the COMP coiled‐coil domain. The retinol β‐ionone ring is positioned in a hydrophobic environment near Thr40, and the 1.1 nm long isoprene tail follows a completely hydrophobic region of the pore. Its terminal hydroxyl group complexes with a ring of the five side chains of Gln54. A mutant in which Gln54 is replaced by Ile binds all‐trans retinol with affinity similar to the wild‐type, demonstrating that hydrophobic interactions are predominant.

The trimer-of-hairpins motif in membrane fusion: Visna virus

Structural studies of viral membrane fusion proteins suggest that a “trimer-of-hairpins” motif plays a critical role in the membrane fusion process of many enveloped viruses. In this motif, a coiled coil (formed by homotrimeric association of the N-terminal regions of the protein) is surrounded by three C-terminal regions that pack against the coiled coil in an oblique antiparallel manner. The resulting trimer-of-hairpins structure serves to bring the viral and cellular membranes together for fusion. learncoil-vmf, a computational program developed to recognize coiled coil-like regions that form the trimer-of-hairpins motif, predicts these regions in the membrane fusion protein of the Visna virus. Peptides corresponding to the computationally identified sequences were synthesized, and the soluble core of the Visna membrane fusion protein was reconstituted in solution. Its crystal structure at 1.5-Å resolution demonstrates that a trimer-of-hairpins structure is formed. Remarkably, despite less than 23% sequence identity, the ectodomains in Visna and HIV-1 envelope glycoproteins show detailed structural conservation, especially within the area of a hydrophobic pocket in the central coiled coil currently being targeted for the development of new anti-HIV drugs.

Conversion of aspartate aminotransferase into an L-aspartate beta-decarboxylase by a triple active-site mutation.

The conjoint substitution of three active-site residues in aspartate aminotransferase (AspAT) of Escherichia coli (Y225R/R292K/R386A) increases the ratio ofl-aspartate β-decarboxylase activity to transaminase activity >25 million-fold. This result was achieved by combining an arginine shift mutation (Y225R/R386A) with a conservative substitution of a substrate-binding residue (R292K). In the wild-type enzyme, Arg386 interacts with the α-carboxylate group of the substrate and is one of the four residues that are invariant in all aminotransferases; Tyr225 is in its vicinity, forming a hydrogen bond with O-3′ of the cofactor; and Arg292interacts with the distal carboxylate group of the substrate. In the triple-mutant enzyme, k cat′ for β-decarboxylation of l-aspartate was 0.08 s−1, whereas k cat′ for transamination was decreased to 0.01 s−1. AspAT was thus converted into an l-aspartate β-decarboxylase that catalyzes transamination as a side reaction. The major pathway of β-decarboxylation directly produces l-alanine without intermediary formation of pyruvate. The various single- or double-mutant AspATs corresponding to the triple-mutant enzyme showed, with the exception of AspAT Y225R/R386A, no measurable or only very low β-decarboxylase activity. The arginine shift mutation Y225R/R386A elicits β-decarboxylase activity, whereas the R292K substitution suppresses transaminase activity. The reaction specificity of the triple-mutant enzyme is thus achieved in the same way as that of wild-type pyridoxal 5′-phosphate-dependent enzymes in general and possibly of many other enzymes, i.e. by accelerating the specific reaction and suppressing potential side reactions.

The three‐dimensional structure of Escherichia coli porphobilinogen deaminase at 1.76‐Å resolution

Porphobilinogen deaminase (PBGD) catalyses the polymerization of four molecules of porphobilinogen to form the 1‐hydroxymethylbilane, preuroporphyrinogen, a key intermediate in the biosynthesis of tetrapyrroles. The three‐dimensional structure of wild‐type PBGD from Escherichia coli has been determined by multiple isomorphous replacement and refined to a crystallographic R‐factor of 0.188 at 1.76 Å resolution. The polypeptide chain of PBGD is folded into three α/β domains. Domains 1 and 2 have a similar overall topology, based on a five‐stranded, mixed β‐sheet. These two domains, which are linked by two hinge segments but otherwise make few direct interactions, form an extensive active site cleft at their interface. Domain 3, an open‐faced, anti‐parallel sheet of three strands, interacts approximately equally with the other two domains. The dipyrromethane cofactor is covalently attached to a cysteine side‐chain borne on a flexible loop of domain 3. The cofactor serves as a primer for the assembly of the tetrapyrrole product and is held within the active site cleft by hydrogen‐bonds and salt‐bridges that are formed between its acetate and propionate side‐groups and the polypeptide chain. The structure of a variant of PBGD, in which the methionines have been replaced with selenomethionines, has also been determined. The cofactor, in the native and functional form of the enzyme, adopts a conformation in which the second pyrrole ring (C2) occupies an internal position in the active site cleft. On oxidation, however, this C2 ring of the cofactor adopts a more external position that may correspond approximately to the site of substrate binding and polypyrrole chain elongation. The side‐chain of Asp84 hydrogen‐bonds the hydrogen atoms of both cofactor pyrrole nitrogens and also potentially the hydrogen atom of the pyrrole nitrogen of the porphobilinogen molecule bound to the proposed substrate binding site. This group has a key catalytic role, possibly in stabilizing the positive charges that develop on the pyrrole nitrogens during the ring‐coupling reactions. Possible mechanisms for the processive elongation of the polypyrrole chain involve: accommodation of the elongating chain within the active site cleft, coupled with shifts in the relative positions of domains 1 and 2 to carry the terminal ring into the appropriate position at the catalytic site; or sequential translocation of the elongating polypyrrole chain, attached to the cofactor on domain 3, through the active site cleft by the progressive movement of domain 3 with respect to domains 1 and 2. Other mechanisms are considered although the amino acid sequence comparisons between PBGDs from all species suggest they share the same three‐dimensional structure and mechanism of activity.