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L. A. Kohlstaedt

Bio: L. A. Kohlstaedt is an academic researcher from Yale University. The author has contributed to research in topics: Reverse transcriptase & RNA-Directed DNA Polymerase. The author has an hindex of 5, co-authored 5 publications receiving 2467 citations.

Papers
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Journal ArticleDOI
26 Jun 1992-Science
TL;DR: 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.
Abstract: 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.

1,902 citations

Journal ArticleDOI
TL;DR: The co-crystal-structure of HIV-1 RT and Nevirapine has been solved previously at 3.5-A resolution and now is partially refined against data extending to 2.9-A spacing, implying that there may be limitations on the number of resistance mutations that yield viable virus.
Abstract: The dipyridodiazepinone Nevirapine is a potent and highly specific inhibitor of the reverse transcriptase (RT) from human immunodeficiency virus type 1 (HIV-1). It is a member of an important class of nonnucleoside drugs that appear to share part or all of the same binding site on the enzyme but are susceptible to a variety of spontaneous drug-resistance mutations. The co-crystal-structure of HIV-1 RT and Nevirapine has been solved previously at 3.5-A resolution and now is partially refined against data extending to 2.9-A spacing. The drug is bound in a hydrophobic pocket and in contact with some 38 protein atoms from the p66 palm and thumb subdomains. Most, but not all, nonnucleoside drug-resistance mutations map to residues in close contact with Nevirapine. The major effects of these mutations are to introduce steric clashes with the drug molecule or to remove favorable protein-drug contacts. Additionally, four residues (Phe-227, Trp-229, Leu-234, and Tyr-319) in contact with Nevirapine have not been selected as sites of drug-resistance mutations, implying that there may be limitations on the number and types of resistance mutations that yield viable virus. Strategies of inhibitor design that target interactions with these conserved residues may yield drugs that are less vulnerable to escape mutations.

328 citations

Journal ArticleDOI
TL;DR: It is suggested that both p51 and p66 form asymmetric homodimers that are assembled from one subunit that has assumed the open conformation and one that has the closed structure, as seen in the p51 subunit of the 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 contains both a polymerase and an RNase H domain; proteolytic cleavage of p66 removes the RNase H domain to yield the p51 subunit. Although the polymerase domain of p66 folds into an open, extended structure containing a large active-site cleft, that of p51 is closed and compact. The connection subdomain, which lies between the polymerase and RNase H active sites in p66, plays a central role in the formation of the reverse transcriptase heterodimer. Extensive and very different intra- and intersubunit contacts are made by the connection subdomains of each of the subunits. Together, contacts between the two connection domains constitute approximately one-third of the total contacts between subunits of the heterodimer. Conversion of an open p66 polymerase domain structure to a closed p51-like structure results in a reduction in solvent-accessible surface area by 1600 A2 and the burying of an extensive hydrophobic surface. Thus, the monomeric forms of both p66 and p51 are proposed to have the same closed structure as seen in the p51 subunit of the heterodimer. The free energy required to convert p66 from a closed p51-like structure to the observed open p66 polymerase domain structure is generated by the burying of a large, predominantly hydrophobic surface area upon formation of the heterodimer. It is likely that the only kind of dimer that can form is an asymmetric one like that seen in the heterodimer structure, since one dimer interaction surface exists only in p51 and the other only in p66. We suggest that both p51 and p66 form asymmetric homodimers that are assembled from one subunit that has assumed the open conformation and one that has the closed structure.

177 citations

Journal ArticleDOI
TL;DR: It is shown that HIV RT can use either tRNA(3Lys) or t RNA(2Gln) as a primer for DNA synthesis in vitro without the addition of any other host or viral proteins.
Abstract: Although the reverse transcriptase (RT) of human immunodeficiency virus (HIV) uses human tRNA(3Lys) as a primer of viral genome DNA synthesis in vivo, HIV RT binds Escherichia coli glutamine tRNA and in vitro-made human lysine tRNA with nearly equivalent affinities. We show that HIV RT can use either tRNA(3Lys) or tRNA(2Gln) as a primer for DNA synthesis in vitro without the addition of any other host or viral proteins. E. coli tRNA(2Gln) can serve as a primer for HIV RT if a primer-binding site sequence complementary to the 3' end of tRNA(2Gln) is at the 3' end of the template. With this reduced template, the specificity of binding the proper tRNA is due to base-pairing between a bound tRNA to the primer-binding site of the viral RNA template rather than sequence-specific recognition of tRNA(3Lys) by RT. If an 8-nucleotide viral sequence 3' to the primer-binding site is included in the template, then addition of Zn2+ or Co2+ is required for tRNA(3Lys)-primed synthesis, and tRNA(2Gln) now fails to prime synthesis. The latter result implies that a template sequence adjacent to the primer-binding site and containing 6 nucleotides complementary to the anticodon loop of human tRNA(3Lys) plays an active role in tRNA discrimination.

72 citations

Journal ArticleDOI
TL;DR: The structure of HIV RT is expected to facilitate the design of new inhibitors that might prove to be effective in the control of AIDS and many laboratories have attempted with varying degrees of success to grow crystals of RT suitable for high-resolution structural analysis.
Abstract: Although reverse transcriptase (RT) was first described in Rous sarcoma virus (RSV) (Baltimore 1970; Temin and Mitzutani 1970), and RSV RT and other RTs from DNA tumor viruses have been studied for many years (Weiss et al. 1982), the recent intense interest in human immunodeficiency virus type 1 (HIV-1) has led to the crystal structure of HIV RT being solved first. HIV RT is the target of 3′-azidodeoxy-thymidine (AZT) and dideoxyinosine (ddI), which are anti-AIDS drugs that function by terminating the DNA during its synthesis (Mitsuya et al. 1990). In addition, a class of nonnucleotide inhibitors of RT also show potential to be less toxic (Merluzzi et al. 1990; Pauwels et al. 1990). The effectiveness of all current compounds in the treatment of AIDS is limited by the toxicity of nucleotide analogs and by mutations in the RT that render it insensitive to both classes of these inhibitors (Larder and Kemp 1989; Shih et al. 1991; St. Clair et al. 1991). Because the structure of HIV RT is expected to facilitate the design of new inhibitors that might prove to be effective in the control of AIDS, many laboratories have attempted with varying degrees of success to grow crystals of RT suitable for high-resolution structural analysis (Lowe et al. 1988; Unge et al. 1990; Jacobo-Molina et al. 1991; Lloyd et al. 1991). Recently, a 3.5-A-resolution crystal structure of RT complexed with a nonnucleotide inhibitor derived from cocrystals that diffract to 3.1-A resolution has been reported (Kohlstaedt et al. 1992). Additionally,...

26 citations


Cited by
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Journal ArticleDOI
TL;DR: GOLD (Genetic Optimisation for Ligand Docking) is an automated ligand docking program that uses a genetic algorithm to explore the full range of ligand conformational flexibility with partial flexibility of the protein, and satisfies the fundamental requirement that the ligand must displace loosely bound water on binding.

5,882 citations

Journal ArticleDOI
18 Jun 1998-Nature
TL;DR: The structure reveals a cavity-laden CD4–gp120 interface, a conserved binding site for the chemokine receptor, evidence for a conformational change upon CD4 binding, the nature of a CD4-induced antibody epitope, and specific mechanisms for immune evasion.
Abstract: The entry of human immunodeficiency virus (HIV) into cells requires the sequential interaction of the viral exterior envelope glycoprotein, gp120, with the CD4 glycoprotein and a chemokine receptor on the cell surface. These interactions initiate a fusion of the viral and cellular membranes. Although gp120 can elicit virus-neutralizing antibodies, HIV eludes the immune system. We have solved the X-ray crystal structure at 2.5 A resolution of an HIV-1 gp120 core complexed with a two-domain fragment of human CD4 and an antigen-binding fragment of a neutralizing antibody that blocks chemokine-receptor binding. The structure reveals a cavity-laden CD4-gp120 interface, a conserved binding site for the chemokine receptor, evidence for a conformational change upon CD4 binding, the nature of a CD4-induced antibody epitope, and specific mechanisms for immune evasion. Our results provide a framework for understanding the complex biology of HIV entry into cells and should guide efforts to intervene.

3,047 citations

Journal ArticleDOI
15 Aug 1997-Science
TL;DR: In this paper, the homologous genes from the fission yeast Schizosaccharomyces pombe and human are identified and the proposed telomerase catalytic subunits represent a deep branch in the evolution of reverse transcriptases.
Abstract: Catalytic protein subunits of telomerase from the ciliate Euplotes aediculatus and the yeast Saccharomyces cerevisiae contain reverse transcriptase motifs. Here the homologous genes from the fission yeast Schizosaccharomyces pombe and human are identified. Disruption of the S. pombe gene resulted in telomere shortening and senescence, and expression of mRNA from the human gene correlated with telomerase activity in cell lines. Sequence comparisons placed the telomerase proteins in the reverse transcriptase family but revealed hallmarks that distinguish them from retroviral and retrotransposon relatives. Thus, the proposed telomerase catalytic subunits are phylogenetically conserved and represent a deep branch in the evolution of reverse transcriptases.

2,181 citations

Journal ArticleDOI
TL;DR: Application of the method is illustrated by the restoration of a ribosome-like model structure and more realistically by the determination of the shape of several proteins from experimental x-ray scattering data.

2,105 citations

Journal ArticleDOI
26 Jun 1992-Science
TL;DR: 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.
Abstract: 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.

1,902 citations