2.2 å resolution structure of the amino-terminal half of HIV-1 reverse transcriptase (fingers and palm subdomains)

Unge, Torsten; Knight, Stefan D.; Bhikhabhai, Ramagauri; Lövgren, Seved; Dauter, Zbigniew; Wilson, K.S.; Strandberg, B.

doi:10.1016/s0969-2126(94)00097-2

Cited by 51 publications

(47 citation statements)

References 20 publications

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“…We believe this to be unlikely, as several of the RT fragments utilized in our studies (FP, FPT, T, TCR, Conn-R, C*R, and R) have led to the reconstitution of a functional enzyme when used in mixing experiments (29). For example, when the Conn-R domain alone or the TC fragment was added to the enzymatically inactive C*R fragment, it resulted in reactivation of R. Furthermore, Unge et al crystallized the FP domain from amino acid residues 1 to 216 and found it to be very close to the structure of the FP domain observed in p66 (34). Similarly, the crystal structure of a fragment of HIV-1 RT corresponding to the R domain is largely comparable to the structure of this domain when the entire heterodimer was crystallized (7,16).…”

Section: Discussionmentioning

confidence: 95%

Interaction between Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Integrase Proteins

Hehl¹,

Joshi²,

Kalpana

et al. 2004

J Virol

102

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Reverse transcription is a key event in the replication cycle of human immunodeficiency virus type 1 (HIV-1) that is initiated after the virus enters the cell. Events subsequent to reverse transcription include nuclear transport of the preintegration complex (PIC), targeting PICs to the sites of integration, proviral integration, and finally the repair of the termini by mechanisms not yet fully understood. Reverse transcription is initiated in the cytoplasm, and the viral cDNA is synthesized in the cytoplasmic compartment. However, it is unclear if reverse transcription is completed prior to proviral integration, as proviral DNA containing discontinuities in plus-strand DNA has been shown to efficiently integrate in vitro. Indeed, reverse transcriptase (RT) has been detected in PICs and intracellular reverse-transcription complexes isolated from the nuclei of HIV-1-infected cells (5, 11). Thus, it is possible that RT is required for polymerization subsequent to nuclear transport, including the completion of plus-strand DNA synthesis and for repair of the single-strand gaps that remain after integration. In certain retrotransposons, exemplified by R2Bm, reverse transcription begins after endonucleolytic cleavage of the genomic DNA by integrase (IN). The RNA is copied using the 3Ј-OH terminus generated by cleavage of the genomic DNA (24), which would necessitate the retention of RT in the nuclear PICs.Furthermore, in avian leukosis virus (14, 30) and in human T-lymphotropic leukemia virus type 1 (33), RT and IN are parts of a single polypeptide forming the ␣ subunit of the heterodimeric ␣␤ complex. In Rous sarcoma virus, where it has been well studied, the ␣␤ complex retains both RT and IN activities. In addition, the recent demonstration that the ␣␤ complex localizes to the nucleus suggests that the RT and IN proteins are likely present together in the nuclear PICs (37). In contrast, the RT and IN proteins of murine retroviruses and of HIV are fully separated by proteolytic cleavage during virion maturation (27). The observation that RT is present in the nuclear PICs (5) in addition to within reverse-transcription complexes (11) suggests that RT may be retained via proteinprotein or protein-nucleic acid interactions with other viral components.Previous studies led to the observation that the RT and IN proteins specifically interact with each other and that this interaction is not mediated by nucleic acid bridging (38). Here, we demonstrate that both monomeric and heterodimeric forms of RT can interact with IN, and we map the domains of interaction on both protein partners. Attempts to assess the effect of IN on RT function showed that RT function was unaffected by IN. In contrast, in the presence of RT, the IN-mediated joining reaction was stimulated significantly, while the 3Ј-end processing was unaffected. These results suggest functional interaction of the two proteins.

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Section: Discussionmentioning

confidence: 95%

Interaction between Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Integrase Proteins

Hehl¹,

Joshi²,

Kalpana

et al. 2004

J Virol

102

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“…This domain structure has been present for all reported crystal forms, but substantial domain rearrangements have been observed [an RT/Fab/DNA complex solved at 3.0 A resolution (Jacobo-Molina et al, 1993); RT complexed with NNIs other than nevirapine at 2.8-3.0 A resolution Ding, Das, Tantillo et al, 1995;Das et al, 1996) and unliganded RTat both 3.2 A resolution (Rodgers et al, 1995) and 2.7 resolution ]. Higher resolution data for fragments of the heterodimer confirm the structure within specific domains [the RNase H domain at 2.4 resolution (Davies et al, 1991) and the fingers and palm domains at 2.2 A resolution (Unge et al, 1994)]. …”

Section: Introductionmentioning

confidence: 84%

Continuous and Discontinuous Changes in the Unit Cell of HIV-1 Reverse Transcriptase Crystals on Dehydration

et al. 1998

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A crystal form of HIV-1 reverse transcriptase (RT) complexed with inhibitors showed diffraction to a highresolution limit of 3.7 ,~. Instability in the unit-cell dimensions of these crystals was observed during soaking experiments, but the range of this variability and consequent change in lattice order was revealed by a chance observation of dehydration. Deliberately induced dehydration results in crystals having a variety of unit cells, the best-ordered of which show diffraction to a minimum Bragg spacing of 2.2,~. In order to understand the molecular basis for this phenomenon, the initial observation of dehydration, the data sets from dehydrated crystals, the crystal packing and the domain conformation of RT are analysed in detail here. This analysis reveals that the crystals undergo remarkable changes following a variety of possible dehydration pathways: some changes occur gradually whilst others are abrupt and require significant domain rearrangements. Comparison of domain arrangements in different crystal forms gives insight into the flexibility of RT which, in turn, may reflect the internal motions allowing this therapeutically important enzyme to fulfill its biological function.

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“…The computer program RIBBONS (13) was used to create the crystal structure figures. The coordinates of the crystal structures used in the analyses were obtained from the Brookhaven Protein Data Bank [1har (14), 1rtj (15), 1hmi (16), and 1mml (17)]. …”

Section: Methodsmentioning

confidence: 99%

A genetic approach for identifying critical residues in the fingers and palm subdomains of HIV-1 reverse transcriptase

Wrobel

Chao

Conrad

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

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By using oligonucleotide-directed saturation mutagenesis, we collected 366 different single amino acid substitutions in a 109-aa segment (residues 95-203) in the fingers and palm subdomains of the HIV-1 reverse transcriptase (RT), the enzyme that replicates the viral genome. After expression in Escherichia coli, two phenotypic assays were performed. The first assay tested for RNA-dependent DNA polymerase activity. The other assay used Western blot analysis to estimate the stability of each mutant protein by measuring the processing of the RT into its mature heterodimeric form, consisting of a 66-kDa subunit and a 51-kDa subunit. The resulting phenotypic data provided a ''genetic'' means to identify amino acid side chains that are important for protein function or stability, as well as side chains located on the protein surface. Several HIV-1 RT crystal structures were used to evaluate the mutational analysis. Our genetic map correlates well with the crystal structures. Combining our phenotype data with crystallographic data allowed us to study the genetically defined critical residues. The important functional residues are found near the enzyme active site. Many residues important for the stability of the RT participate in potential hydrogen bonding or hydrophobic interactions in the protein interior. In addition to providing a better understanding of the HIV-1 RT, this work demonstrates the utility of saturation mutagenesis to study the function, structure, and stability of proteins in general. This strategy should be useful for studying proteins for which no crystallographic data are available.The development of oligonucleotide-directed mutagenesis (1) enhanced the power of genetic analysis by providing a method to introduce a mutation at a specific position in a DNA sequence. This technique may be used to make a specific amino acid substitution in a protein by altering codons. An adaptation of site-specific mutagenesis led to saturation mutagenesis (reviewed in ref. 2), which provides an approach to make libraries containing a collection of mutations at every base in a DNA element (3) or at every amino acid in a protein (4).X-ray crystallography is a powerful tool for studying protein function and structure. However, the use of this method is limited to proteins that crystallize. With the constant discovery of new proteins with biological and medical importance, additional methods that provide functional and structural information are extremely valuable. In this study, we assess the potential of saturation mutagenesis for the study of function and stability of proteins for which structural data are difficult to obtain, and as a means to offer additional insight into proteins whose structures are known. By using saturation mutagenesis we collected 366 unique single missense mutants in a 109-aa region (residues P95 through E203) in the fingers and palm subdomains of the HIV-1 reverse transcriptase (RT).The HIV-1 RT is encoded by the viral pol gene, which contains the coding sequences for the protease, R...

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2.2 å resolution structure of the amino-terminal half of HIV-1 reverse transcriptase (fingers and palm subdomains)

Cited by 51 publications

References 20 publications

Interaction between Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Integrase Proteins

Interaction between Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Integrase Proteins

Continuous and Discontinuous Changes in the Unit Cell of HIV-1 Reverse Transcriptase Crystals on Dehydration

A genetic approach for identifying critical residues in the fingers and palm subdomains of HIV-1 reverse transcriptase

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