A 3.5 angstrom resolution electron density map of the HIV-1 reverse transcriptase heterodimer complexed with nevirapine, a drug with potential for treatment of AIDS, reveals an asymmetric dimer. The polymerase (pol) domain of the 66-kilodalton subunit has a large cleft analogous to that of the Klenow fragment of Escherichia coli DNA polymerase I. However, the 51-kilodalton subunit of identical sequence has no such cleft because the four subdomains of the pol domain occupy completely different relative positions. Two of the four pol subdomains appear to be structurally related to subdomains of the Klenow fragment, including one containing the catalytic site. The subdomain that appears likely to bind the template strand at the pol active site has a different structure in the two polymerases. Duplex A-form RNA-DNA hybrid can be model-built into the cleft that runs between the ribonuclease H and pol active sites. Nevirapine is almost completely buried in a pocket near but not overlapping with the pol active site. Residues whose mutation results in drug resistance have been approximately located.

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High-resolution crystal structures of editing complexes of both duplex and single-stranded DNA bound to Escherichia coli DNA polymerase I large fragment (Klenow fragment) show four nucleotides of single-stranded DNA bound to the 3'-5' exonuclease active site and extending toward the polymerase active site. Melting ofthe duplex DNA by the protein is stabilized by hydophobic interactions between Phe473, Leu-361, and His-666 and the last three bases at the 3' terminus. Two divalent metal ions interacting with the phosphodiester to be hydrolyzed are proposed to catalyze the exonuclease reaction by a mechanism that may be related to mechanisms of other enzymes that catalyze phospho-group transfer including RNA enzymes. We suggest that the editing active site competes with the polymerase active site some 30 A away for the newly formed 3' terminus. Since a 3' terminal mismatched base pair favors the melting of duplex DNA, its binding and excision at the editing exonuclease site that binds single-stranded DNA is enhanced.The large proteolytic fragment (Klenow fragment) of Escherichia coli DNA polymerase I utilizes an editing 3'-5' exonuclease activity (1) to reduce the misincorporation of erroneous nucleotides by about 10-fold (2) at an active site that is some 30 A away from the polymerase site of misincorporation (3). How might this be accomplished? The crystal structure of the Klenow fragment shows that it is folded into two domains (3). Various experiments (reviewed in ref. 4) establish that the domain to which the dNMP binds in the crystal catalyzes the 3'-5' econuclease activity, whereas the larger C-terminal domain contains the active site for the polymerase reaction. Mutant proteins that contain amino acid changes in the dNMP binding site have been made by directed mutagenesis; they are devoid of exonuclease activity but retain full polymerase activity (5). Furthermore, the DNA encoding the C-terminal domain has been cloned, and the product has been expressed, isolated, and shown to possess significant DNA polymerase activity with no measurable 3'-5' exonuclease activity (6). The observation (3) that these two active sites are -25-30 A apart poses the interesting question of how they work together to achieve high-fidelity synthesis of DNA.The C-terminal domain contains a cleft that is large enough to accommodate the double-stranded B-DNA product of DNA synthesis (3). The approximate position of the 3' terminus of the primer strand has been derived from the cross-linking of 8-azido-dATP to Tyr-776, footprinting of Klenow fragment on DNA (7), and the position of sitedirected mutants that alter polymerase activity but not exonuclease activity (A. Polesky and C. Joyce, personal communication). This model of DNA at the polymerase active site places about 8 base pairs (bp) of duplex product DNA in the cleft.A more detailed understanding of the structural basis ofthe polymerase and exonuclease activities requires the separate determination of the crystal structures of suitable DNAs complexed with each of these ...

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...

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Space-filling models of yeast hexokinase, adenylate kinase, and phosphoglycerate kinase drawn by computer clearly portray the bilobal character of these phosphoryl transfer enzymes, and the deep cleft which is formed between the lobes. A dramatic conformational change occurs in hexokinase as glucose binds to the bottom of the cleft, which causes the two lobes of hexokinase to come together. A substrate-induced closing of the active site cleft is postulated to occur in other kinases as well. This change may provide a mechanism by which some of these enzymes reduce their inherent adenosine triphosphatase activity and could be a general requirement of the kinase reaction.

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The packing arrangement of the 12 subunits of intact gamma delta resolvase in the unit cell of a hexagonal crystal form suggests a model for site‐specific recombination that involves a DNA‐mediated synaptic intermediate. The crystal structure has been determined by molecular replacement and partially refined at 2.8/3.5 A resolution. Although the small DNA‐binding domain is disordered in these crystals, packing considerations show that only a small region of space in the crystal could accommodate a domain of its size. A family of related models for a synaptic complex between two DNA duplexes and 12 monomers that are arranged as situated in the crystal is consistent with the known topology of the complex and the distances between the three resolvase dimer‐binding sites per DNA; further, these models place the two DNA recombination sites in contact with each other between two resolvase dimers, implying that strand exchange is accomplished through direct DNA‐DNA interaction. A major role postulated, then, for the resolvase protein assembly is to stabilize a res DNA structure that is close to the topological transition state of the reaction.