Marie L. Coté

Murine Leukemia Virus Reverse Transcriptase: Structural Comparison with HIV-1 Reverse Transcriptase

Recent X-ray crystal structure determinations of Moloney murine leukemia virus reverse transcriptase (MoMLV RT) have allowed for more accurate structure/function comparisons to HIV-1 RT than were formerly possible. Previous biochemical studies of MoMLV RT in conjunction with knowledge of sequence homologies to HIV-1 RT and overall fold similarities to RTs in general, provided a foundation upon which to build. In addition, numerous crystal structures of the MoMLV RT fingers/palm subdomain had also shed light on one of the critical functions of the enzyme, specifically polymerization. Now in the advent of new structural information, more intricate examination of MoMLV RT in its entirety can be realized, and thus the comparisons with HIV-1 RT may be more critically elucidated. Here, we will review the similarities and differences between MoMLV RT and HIV-1 RT via structural analysis, and propose working models for the MoMLV RT based upon that information.

Crystal structure of the DNA-binding domain from Ndt80, a transcriptional activator required for meiosis in yeast

Ndt80 is a transcriptional activator required for meiosis in the yeast Saccharomyces cerevisiae. Here, we report the crystal structure at 2.3 Å resolution of the DNA-binding domain of Ndt80 experimentally phased by using the anomalous and isomorphous signal from a single ordered Se atom per molecule of 272-aa residues. The structure reveals a single ≈32-kDa domain with a distinct fold comprising a β-sandwich core elaborated with seven additional β-sheets and three short α-helices. Inspired by the structure, we have performed a mutational analysis and defined a DNA-binding motif in this domain. The DNA-binding domain of Ndt80 is homologous to a number of proteins from higher eukaryotes, and the residues that we have shown are required for DNA binding by Ndt80 are highly conserved among this group of proteins. These results suggest that Ndt80 is the defining member of a previously uncharacterized family of transcription factors, including the human protein (C11orf9), which has been shown to be highly expressed in invasive or metastatic tumor cells.

Revealing Domain Structure through Linker-Scanning Analysis of the Murine Leukemia Virus (MuLV) RNase H and MuLV and Human Immunodeficiency Virus Type 1 Integrase Proteins

ABSTRACT Linker-scanning libraries were generated within the 3′ terminus of the Moloney murine leukemia virus (M-MuLV) pol gene encoding the connection-RNase H domains of reverse transcriptase (RT) as well as the structurally related M-MuLV and human immunodeficiency virus type 1 (HIV-1) integrase (IN) proteins. Mutations within the M-MuLV proviral vectors were Tn7 based and resulted in 15-bp insertions. Mutations within an HIV-1 IN bacterial expression vector were based on Tn5 and resulted in 57-bp insertions. The effects of the insertions were examined in vivo (M-MuLV) and in vitro (HIV-1). A total of 178 individual M-MuLV constructs were analyzed; 40 in-frame insertions within RT connection-RNase H, 108 in-frame insertions within IN, 13 insertions encoding stop codons within RNase H, and 17 insertions encoding stop codons within IN. For HIV-1 IN, 56 mutants were analyzed. In both M-MuLV and HIV-1 IN, regions are identified which functionally tolerate multiple-linker insertions. For MuLV, these correspond to the RT-IN proteolytic junction, the junction between the IN core and C terminus, and the C terminus of IN. For HIV-1 IN, in addition to the junction between the IN core and C terminus and the C terminus of IN, insertions between the N terminus and core domains maintained integration and disintegration activity. Of the 40 in-frame insertions within the M-MuLV RT connection-RNase H domains, only the three C-terminal insertions mapping to the RT-IN proteolytic junction were viable. These results correlate with deletion studies mapping the domain and subdomain boundaries of RT and IN. Importantly, these genetic footprints provide a means to identify nonessential regions within RT and IN for targeted gene therapy applications.

Crystal structures of oligonucleotides including the integrase processing site of the Moloney murine leukemia virus

In the first step of retroviral integration, integrase cleaves the linear viral DNA within its long terminal repeat (LTR) immediately 3′ to the CA dinucleotide step, resulting in a reactive 3′ OH on one strand and a 5′ two base overhang on the complementary strand. In order to investigate the structural properties of the 3′ end processing site within the Moloney murine leukemia virus (MMLV) LTR d(TCTTTCATT), a host-guest crystallographic method was employed to determine the structures of four self-complementary 16 bp oligonucleotides including LTR sequences (underlined), d(TTTCATTGCAATGAAA), d(CTTTCATTAATGAAAG), d(TCTTTCATATGAAAGA) and d(CACAATGATCATTGTG), the guests, complexed with the N-terminal fragment of MMLV reverse transcriptase, the host. The structures of the LTR-containing oligonucleotides were compared to those of non-LTR oligonucleotides crystallized in the same lattice. Properties unique to the CA dinucleotide step within the LTR sequence, independent of its position from the end of the duplex, include a positive roll angle and negative slide value. This propensity for the CA dinucleotide step within the MMLV LTR sequence to adopt only positive roll angles is likely influenced by the more rigid, invariable 3′ and 5′ flanking TT dinucleotide steps and may be important for specific recognition and/or cleavage by the MMLV integrase.

Crystallographic studies of a novel DNA-binding domain from the yeast transcriptional activator Ndt80

The Ndt80 protein is a transcriptional activator that plays a key role in the progression of the meiotic divisions in the yeast Saccharomyces cerevisiae. Ndt80 is strongly induced during the middle stages of the sporulation pathway and binds specifically to a promoter element called the MSE to activate transcription of genes required for the meiotic divisions. Here, the preliminary structural and functional studies to characterize the DNA-binding activity of this protein are reported. Through deletion analysis and limited proteolysis studies of Ndt80, a novel 32 kDa DNA-binding domain that is sufficient for DNA-binding in vitro has been defined. Crystals of the DNA-binding domain of Ndt80 in two distinct lattices have been obtained, for which diffraction data extend to 2.3 A resolution.

Expression of an Mg2+-Dependent HIV-1 RNase H Construct for Drug Screening

ABSTRACT A single polypeptide of the HIV-1 reverse transcriptase that reconstituted Mg2+-dependent RNase H activity has been made. Using molecular modeling, the construct was designed to encode the p51 subunit joined by a linker to the thumb (T), connection (C), and RNase H (R) domains of p66. This p51-G-TCR construct was purified from the soluble fraction of an Escherichia coli strain, MIC2067(DE3), lacking endogenous RNase HI and HII. The p51-G-TCR RNase H construct displayed Mg2+-dependent activity using a fluorescent nonspecific assay and showed the same cleavage pattern as HIV-1 reverse transcriptase (RT) on substrates that mimic the tRNA removal required for second-strand transfer reactions. The mutant E706Q (E478Q in RT) was purified under similar conditions and was not active. The RNase H of the p51-G-TCR RNase H construct and wild type HIV-1 RT had similar Kms for an RNA-DNA hybrid substrate and showed similar inhibition kinetics to two known inhibitors of the HIV-1 RT RNase H.

(+)- and (±)-ketopinic acid : hydrogen-bonding patterns in a β-keto acid in its enantiomeric and racemic forms and enantiomeric disordering in the racemate

Crystal structures have been determined for both (+)-and (±)-7,7-dimethyl-2-oxobicyclo[2.2. 1 ]heptane- 1 -carboxylic acid, C 10 H 14 O 3 . The asymmetric unit of the (+)-form, (I), contains two molecules paired by mutual hydrogen bonding of their carboxyl groups, without ketone involvement. Both carboxyl groups are disordered and the conformation of each relative to its own molecule differs by 46.3 (4)° for the two halves of the dimer. In the racemate, (II), the asymmetric unit also contains two molecules, each of which dimerizes with a centrosymmetric counterpart by mutual hydrogen bonding of carboxyl groups across the a or b cell edge, also without ketone involvement. Besides disorder of the carboxyl C-O lengths and angles, each half of the asymmetric unit of (II) displays extensive enantiomeric disorder, corresponding to transposition of the ethylene and methylene bridges, with the carboxyl and ketone C atoms plus the anti-methyl C atom exactly coinciding. Each contributor to the structure of (II) has a different rotational conformation for its carboxyl group, none corresponding to those found in (I).

1-Carboxyfluorenone: an Intramolecularly Hydrogen-Bonded γ-Keto Acid

9-Oxo-9H-fluorene-1-carboxylic acid, C 14 H 8 O 3 , adopts a planar conformation with the carboxyl group internally hydrogen bonded to the ketone O atom. The molecules stack in the a direction [a = 3.809 (1)A], with an interplanar separation of 3.496 (4) A.

2-Hydroxyisophthalic acid: hydrogen-bonding patterns in the monohydrate and the tetraphenylphosphonium salt. An instance of dramatic acidity enhancement by symmetric, internally hydrogen-bonded anion stabilization.

The monohydrate of the title phenolic diacid (C8H6O5.H2O, 2-hydroxybenzene-1,3-dioic acid or 3-carboxysalicylic acid) adopts a planar conformation, with the phenol hydrogen internally hydrogen-bonded to the carbonyl of one highly ordered carboxyl, which, in turn, donates a hydrogen bond to the oxygen of water. The second carboxyl is disordered and hydrogen-bonded both to water and to the disordered carboxyl of a centrosymmetrically related neighbor in a static disorder arrangement extending over two full asymmetric units. The water accepts either one or two hydrogen bonds and donates a long bifurcated hydrogen bond shared equally by O atoms of the phenol and the disordered carboxyl. The hydrogen bonding includes no standard carboxyl pairing and is entirely two-dimensional. The resulting planar ribbons stack translationally at a distance of 3.413 (8) A, in an offset arrangement having non-translational interplanar distances of 0.821 (5) and 2.592 (6) A. This structure is compared with two previously reported for this compound. The title compound forms a monoanion, whose tetraphenylphosphonium salt is described (C32H25O5P, tetraphenylphosphonium 2,6-dicarboxyphenolate, tetraphenylphosphonium 2-oxidoisophthalic acid or tetraphenylphosphonium 3-carboxysalicylate). The phenol oxygen is the site of formal negative charge on the anion, which is stabilized in a planar arrangement by symmetrical hydrogen bonds from both ortho-carboxyl groups. The energetics of this arrangement, the phenol and carboxyl acidities, and factors affecting those acidities and providing anion stabilization are discussed.

X‐ray Crystal Structure of the N‐Terminal Region of Moloney Murine Leukemia Virus Integrase and its Implications for Viral DNA Recognition

The retroviral integrase (IN) carries out the integration of a dsDNA copy of the viral genome into the host DNA, an essential step for viral replication. All IN proteins have three general domains, the N‐terminal domain (NTD), the catalytic core domain, and the C‐terminal domain. The NTD includes an HHCC zinc finger‐like motif, which is conserved in all retroviral IN proteins. Two crystal structures of Moloney murine leukemia virus (M‐MuLV) IN N‐terminal region (NTR) constructs that both include an N‐terminal extension domain (NED, residues 1–44) and an HHCC zinc‐finger NTD (residues 45–105), in two crystal forms are reported. The structures of IN NTR constructs encoding residues 1–105 (NTR1–105) and 8–105 (NTR8–105) were determined at 2.7 and 2.15 Å resolution, respectively and belong to different space groups. While both crystal forms have similar protomer structures, NTR1–105 packs as a dimer and NTR8–105 packs as a tetramer in the asymmetric unit. The structure of the NED consists of three anti‐parallel β‐strands and an α‐helix, similar to the NED of prototype foamy virus (PFV) IN. These three β‐strands form an extended β‐sheet with another β‐strand in the HHCC Zn2+ binding domain, which is a unique structural feature for the M‐MuLV IN. The HHCC Zn2+ binding domain structure is similar to that in HIV and PFV INs, with variations within the loop regions. Differences between the PFV and MLV IN NEDs localize at regions identified to interact with the PFV LTR and are compared with established biochemical and virological data for M‐MuLV. Proteins 2017; 85:647–656.