Structural insights into the specific anti-HIV property of actinohivin: structure of its complex with the α(1–2)mannobiose moiety of gp120

Abstract

Actinohivin (AH) is an actinomycete lectin with a potent specific anti-HIV activity. In order to clarify the structural evidence for its specific binding to the (1-2)mannobiose (MB) moiety of the D1 chains of high-mannose-type glycans (HMTGs) attached to HIV-1 gp120, the crystal structure of AH in complex with MB has been determined. The AH molecule is composed of three identical structural modules, each of which has a pocket in which an MB molecule is bound adopting a bracket-shaped conformation. This conformation is stabilized through two weak C-HÁ Á ÁO hydrogen bonds facilitated by the (1-2) linkage. The binding features in the three pockets are quite similar to each other, in accordance with the molecular pseudo-threefold symmetry generated from the three tandem repeats in the amino-acid sequence. The shape of the pocket can accept two neighbouring hydroxyl groups of the O 3 and O 4 atoms of the equatorial configuration of the second mannose residue. To recognize these atoms through hydrogen bonds, an Asp residue is located at the bottom of each pocket. Tyr and Leu residues seem to block the movement of the MB molecules. Furthermore, the O 1 atom of the axial configuration of the second mannose residue protrudes from each pocket into an open space surrounded by the conserved hydrophobic residues, suggesting an additional binding site for the third mannose residue of the branched D1 chain of HMTGs. These structural features provide strong evidence indicating that AH is only highly specific for MB and would facilitate the highly specific affinity of AH for any glycoprotein carrying many HMTGs, such as HIV-1 gp120.

The Characteristic Structure of Anti‐HIV Actinohivin in Complex with Three HMTG D1 Chains of HIV‐gp120

“…The crystal structure was determined at 1.40 resolution with low R factor (R free ) values-0.14 (0.17)-so the electron density distribution shows a well-resolved atomic structure of AH, in addition to which two mannose residues of bound D1(a(1,2)mannotriose) molecules are also well resolved in each of the three modules 1 of AH, as shown in Figure 1 A. In the previous reports, [14,15] we postulated the two pockets I and II for the D1 and D3 chain bindings in each module. Two mannose residues of D1 are clearly bound in pocket I of the modules.…”

“…Local superimpositions between Man1 and Man2 and between Man2 and Man3 show low rmsd values from 0.20 to 0.29 , thus suggesting that the two a(1,2) linkages are the same bracket-shape conformation similar to those observed in the AH·MB complexes. [14,15] Similarly, superimpositions of six D1s give low rmsd values from 0.31 to 0.89 , thus showing that D1 adopts a Figure 1. j F o j À j F c j omit maps of A) the two mannose residues bound in zones 1 and 2, and B) the remaining zones 3 and 0, of module 2.…”

“…Such water-mediated recognition is also found in the AH·MB complexes. [14,15] The most striking feature is found in the interaction of the Man1 and Man3 residues with AH. As shown in Figure 5 A and B, all eight hydroxy groups of the two Man residues are completely exposed in the solvent region, so that they have no direct contacts with amino acid residues of AH.…”

“…A) Overall structure of AH in complex with three D1 molecules, and B) the two modes. In (A) I and II suggest the two pockets proposed for the D1 and D3 chain binding modes, [11,14] and a triangle with three dotted lines, which shows the molecular pseudo-threefold symmetry, divides the three modules 1, 2, and 3, schematically. In (B), zone 0 means outside of the AH molecule, and zones 1-3 show the three mannose-residue-binding zones in each pocket I.…”

“…In order to obtain structural insights into AH, which specifically binds to this moiety, the crystal structures of AH in the targetfree state [12] and AHs in complex with MBs [14,15] were determined by X-ray analysis. However, the three branched chains exhibit different binding affinities, it being estimated that that of D1 is the strongest, that of D3 is weaker, and that of D2 is undetectable, and this is ascribed to the differences in the linkages between the second and the third mannose residues.…”