Structural basis of asymmetry in the human immunodeficiency virus type 1 reverse transcriptase heterodimer.

Abstract

The reverse transcriptase from human immunodeficiency virus type 1 is a heterodimer consisting of one 66-kDa and one 51-kDa subunit. The p66 subunit Perhaps the most surprising aspect of the structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is the observation that the polymerase domain assumes a different structure in the two subunits in spite of having the same polypeptide chain sequence (1). HIV-1 RT consists of one 66-kDa polypeptide chain (p66) consisting of a polymerase domain and an RNase H domain and one 51-kDa polypeptide chain (p51) containing only the polymerase domain. These two subunits interact asymmetrically to generate only one polymerase cleft that binds one primer-template, one dNTP, one noncompetitive inhibitor, and one tRNA (2-5). The polymerase domains of p51 and p66 differ by having an alternative arrangement of four subdomains (1). The three subdomains that form the large polymerase active-site cleft in p66 are called "fingers," "thumb," and "palm" by analogy ofthis polymerase structure to that ofa right hand. The fourth subdomain is called "connection" because it lies between the polymerase and RNase H active sites in p66. Although the heterodimer is the most stable dimer, with an equilibrium dissociation constant (Kd) of =1 x 10-9 M (6, 7), both p66 and p51 homodimers have been observed in vitro but are much less tightly associated (7). The major question addressed here is how a single amino acid sequence can form two quite different structures and result in such an asymmetric subunit interaction. Furthermore, and in the light of the structural observations, it is of interest to consider the likely conformation of pSi or p66 monomers as well as the possible structures of homodimers of these subunits.The crystal structure of the HIV-1 RT heterodimer complexed with a noncompetitive inhibitor, Nevirapine, was initially derived from a 3.5-A resolution electron density map (1) and has now been partially refined at 2.9-A resolution (8, 9). The structure of HIV-1 RT complexed with the Fab portion of a monoclonal antibody and duplex DNA determined at 3-A resolution shows the same structure for the RT and provides experimental evidence for the primer-template location (10). RESULTS AND DISCUSSIONThe Asymmetric Dimer Structure. The four polymerase subdomains of HIV-1 RT have very different relative orientations in the two subunits ofthe RT heterodimer (Fig. 1). The p51 subunit has a compact structure that we can refer to as "closed," while the p66 subunit has a more extended structure and a large cleft that can be referred to as "open." With the connection domains oriented identically, the different sets of interactions made by the fingers, palm, and thumb subdomains of each ofthe two subunits are clearly seen (Fig. 1). Changes in the contacts between the connection and the fingers subdomains are more modest.Interactions between the two subunits are completely asymmetric in that the subunit interface on p51 involves different amino acid residues than the...

TSAO Compounds: The Comprehensive Story of a Unique Family of HIV- 1 Specific Inhibitors of Reverse Transcriptase

“…The abasic TSAO analogue (14) is just one order of magnitude less active than the prototype TSAO-T, and our calculations suggest that this may be due to the energy penalty involved in breaking the intramolecular hydrogen bond in order to adopt a suitable conformation for binding into the enzyme. Our experimental data on TSAO derivatives strongly suggest a specific interaction of the amino group of the 3'-spiro moiety of TSAO molecules with the carboxylic group of a glutamic acid residue at position 138 of the p51 subunit of HIV-1 RT [18,39,40,50,51]. This residue is located at the top of the finger domain of the p51 subunit of HIV-1 RT, that is part of the binding pocket of the HIV-1-specific RT inhibitors at the p66 subunit [16a, 30, 31].…”

“…: (+34)-91 562 29 00; Fax. : (+34)-91 5644853; E-mail: mj.camarasa@iqm.csic.es both subunits, the polymerase subdomains (called fingers, palm, thumb, and connection) are arranged differently in each one, with p66 forming a large active-site cleft and p51 forming an inactive closed structure that provides structural support to the polymerase domain of p66 [18][19][20]. The catalytic site (located at this subunit) where polymerization occurs contains a triad of aspartic acid residues at positions 110, 185 and 186 [21].…”

Phylogenetic Analysis of Ribonuclease H Domains Suggests a Late, Chimeric Origin of LTR Retrotransposable Elements and Retroviruses

“…In the case of the HIV protein, the best studied retroviral reverse transcriptase, the active enzyme is a heterodimer composed of a p66 subunit containing an RT, a connection and an RNH domain and a p51 subunit containing only the RT and connection domains. Several studies have remarked on the structural and possible functional role of the connection domain in the formation of this heterodimer (Wang et al 1994;Divita et al 1994;Debyser and De Clercq 1996). For example, it has been shown to be crucial in mediating the conformational changes required of the p66/p51 heterodimer for reverse transcription (Bahar et al 1999) and for RNH activity (Smith et al 1994).…”

Asymmetric Subunit Organization of Heterodimeric Rous Sarcoma Virus Reverse Transcriptase αβ: Localization of the Polymerase and RNase H Active Sites in the α Subunit

“…The polymerase active site is present only in the larger p66 subunit of the heterodimer (13,14,19,36), while the p51 subunit, which is identical in sequence to p66 but lacks the RNase H domain, is not directly involved in catalysis (21). However, p51 has to fulfill an important stabilizing function since the monomeric subunits are inactive (26,27).…”