Robert Larocque

Structure of the 16S rRNA pseudouridine synthase RsuA bound to uracil and UMP.

In Escherichia coli, the pseudouridine synthase RsuA catalyzes formation of pseudouridine (ψ) at position 516 in 16S rRNA during assembly of the 30S ribosomal subunit. We have determined the crystal structure of RsuA bound to uracil at 2.0 Å resolution and to uridine 5′-monophosphate (UMP) at 2.65 Å resolution. RsuA consists of an N-terminal domain connected by an extended linker to the central and C-terminal domains. Uracil and UMP bind in a cleft between the central and C-terminal domains near the catalytic residue Asp 102. The N-terminal domain shows structural similarity to the ribosomal protein S4. Despite only 15% amino acid identity, the other two domains are structurally similar to those of the tRNA-specific ψ-synthase TruA, including the position of the catalytic Asp. Our results suggest that all four families of pseudouridine synthases share the same fold of their catalytic domain(s) and uracil-binding site.

Constitutive activation of the Saccharomyces cerevislae mating response pathway by a MAP kinase kinase from Candida albicans

The HST7 gene of Candida albicans encodes a protein with structural similarity to MAP kinase kinases. Expression of this gene in Saccharomyces cerevisiae complements disruption of the Ste7 MAP kinase kinase required for both mating in haploid cells and pseudohyphal growth in diploids. However, Hst7 expression does not complement loss of either the Pbs2 (Hog4) MAP kinase kinase required for response to high osmolarity, or loss of the Mkk1 and Mkk2 MAP kinase kinases required for proper cell wall biosynthesis. Intriguingly, HST7 acts as a hyperactive allele of STE7; expression of Hst7 activates the mating pathway even in the absence of upstream signaling components including the Ste7 regulator Ste11, elevates the basal level of the pheromone-inducible FUS1 gene, and amplifies the pseudohyphal growth response in diploid cells. Thus Hst7 appears to be at least partially independent of upstream activators or regulators, but selective in its activity on downstream target MAP kinases. Creation of Hst7/Ste7 hybrid proteins revealed that the C-terminal two-thirds of Hst7, which contains the protein kinase domain, is sufficient to confer this partial independence of upstream activators.

Mechanism of action and NAD+-binding mode revealed by the crystal structure of l-histidinol dehydrogenase

The histidine biosynthetic pathway is an ancient one found in bacteria, archaebacteria, fungi, and plants that converts 5-phosphoribosyl 1-pyrophosphate to l-histidine in 10 enzymatic reactions. This pathway provided a paradigm for the operon, transcriptional regulation of gene expression, and feedback inhibition of a pathway. l-histidinol dehydrogenase (HisD, EC 1.1.1.23) catalyzes the last two steps in the biosynthesis of l-histidine: sequential NAD-dependent oxidations of l-histidinol to l-histidinaldehyde and then to l-histidine. HisD functions as a homodimer and requires the presence of one Zn2+ cation per monomer. We have determined the three-dimensional structure of Escherichia coli HisD in the apo state as well as complexes with substrate, Zn2+, and NAD+ (best resolution is 1.7 Å). Each monomer is made of four domains, whereas the intertwined dimer possibly results from domain swapping. Two domains display a very similar incomplete Rossmann fold that suggests an ancient event of gene duplication. Residues from both monomers form the active site. Zn2+ plays a crucial role in substrate binding but is not directly involved in catalysis. The active site residue His-327 participates in acid-base catalysis, whereas Glu-326 activates a water molecule. NAD+ binds weakly to one of the Rossmann fold domains in a manner different from that previously observed for other proteins having a Rossmann fold.

The solution structure of YbcJ from Escherichia coli reveals a recently discovered alphaL motif involved in RNA binding.

The structure of the recombinant Escherichia coli protein YbcJ, a representative of a conserved family of bacterial proteins (COG2501), was determined by nuclear magnetic resonance. The fold of YbcJ identified it as a member of the larger family of S4-like RNA binding domains. These domains bind to structured RNA, such as that found in tRNA, rRNA, and a pseudoknot of mRNA. The structure of YbcJ revealed a highly conserved patch of basic residues, comprising amino acids K26, K38, R55, K56, and K59, which likely participate in RNA binding.

A structural genomics pilot project based on gene targets selected from Escherichia coli

A pilot project based on gene targets selected from the genome of E. coli has been initiated with 38 genes for initial cloning. Of these, 18 proteins have been purified to date and some crystals were obtained for twelve of them. Of these, four proteins yielded crystals diffracting to a sufficiently high resolution to warrant structural investigation. We have determined 3-D structures of three of these proteins using Se-Met labeling and MAD methods, while the structure of the fourth one was simultaneously determined by another group. To manage the parallel work on many proteins by several researchers it became necessary to create a searchable database containing the pertinent information about every stage of the work.