Li Kai Wang

Structural genomics: a pipeline for providing structures for the biologist.

Progress in understanding the organization and sequences of genes in model organisms and humans is rapidly accelerating. Although genome sequences from prokaryotes have been available for some time, only recently have the genome sequences of several eukaryotic organisms been reported, including Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, and humans (Green 2001). A logical continuation of this line of scientific inquiry is to understand the structure and function of all genes in simple and complex organisms, including the pathways leading to the organization and biochemical function of macromolecular assemblies, organelles, cells, organs, and whole life forms. Such investigations have been variously called integrative or systems biology and -omics or high-throughput biology (Ideker et al. 2001, Greenbaum et al. 2001, Vidal 2001). These studies have blossomed because of advances in technologies that allow highly parallel examination of multiple genes and gene products as well as a vision of biology that is not purely reductionist. Although a unified understanding of biological organisms is still far in the future, new high-throughput biological approaches are having a drastic impact on the scientific mainstream. One offshoot of the high-throughput approach, which directly leverages the accumulating gene sequence information, involves mining the sequence data to detect important evolutionary relationships, to identify the basic set of genes necessary for independent life, and to reveal important metabolic processes in humans and clinically relevant pathogens. Programs such as MAGPIE (www.genomes.rockefeller.edu/magpie/magpie.html) compare organisms at a whole genome level (Gaasterland and Sensen 1996; Gaasterland and Ragan 1998) and ask what functions are conferred by the new genes that have evolved in higher organisms (Gaasterland and Oprea 2001). Concurrent with computational annotations of gene structure and function, thousands of full-length ORFs from yeast and higher eukaryotes have become available because of advances in cloning and other molecular biology techniques (Walhout et al. 2000a). Structural biologists have embraced high-throughput biology by developing and implementing technologies that will enable the structures of hundreds of protein domains to be solved in a relatively short time. Although thousands of structures are deposited annually in the Protein Data Bank (PDB), most are identical or very similar in sequence to a structure previously existing in the data bank, representing structures of mutants or different ligand bound states (Brenner et al. 1997). Providing structural information for a broader range of sequences requires a focused effort on determining structure for sequences that are divergent from those already in the database. Although structure does not always elucidate function, in many instances (including the structures of two proteins reported here) the atomic structure readily provides insight into the function of a protein whose function was previously unknown. Typically, such functional annotations are based on homologies that are not recognizable at the sequence level but that are clearly revealed on inspection of the protein fold, identification of a conserved constellation of side-chain functionalities, or by the observation of cofactors associated with function (Burley et al. 1999; Shi et al. 2001; Bonanno et al. 2002).

Structure and mechanism of T4 polynucleotide kinase: an RNA repair enzyme.

T4 polynucleotide kinase (Pnk), in addition to being an invaluable research tool, exemplifies a family of bifunctional enzymes with 5′‐kinase and 3′‐phosphatase activities that play key roles in RNA and DNA repair. T4 Pnk is a homotetramer composed of a C‐terminal phosphatase domain and an N‐terminal kinase domain. The 2.0 Å crystal structure of the isolated kinase domain highlights a tunnel‐like active site through the heart of the enzyme, with an entrance on the 5′ OH acceptor side that can accommodate a single‐stranded polynucleotide. The active site is composed of essential side chains that coordinate the β phosphate of the NTP donor and the 3′ phosphate of the 5′ OH acceptor, plus a putative general acid that activates the 5′ OH. The structure rationalizes the different specificities of T4 and eukaryotic Pnk and suggests a model for the assembly of the tetramer.

Structure and Mechanism of Yeast RNA Triphosphatase: An Essential Component of the mRNA Capping Apparatus

RNA triphosphatase is an essential mRNA processing enzyme that catalyzes the first step in cap formation. The 2.05 A crystal structure of yeast RNA triphosphatase Cet1p reveals a novel active site fold whereby an eight-stranded beta barrel forms a topologically closed triphosphate tunnel. Interactions of a sulfate in the center of the tunnel with a divalent cation and basic amino acids projecting into the tunnel suggest a catalytic mechanism that is supported by mutational data. Discrete surface domains mediate Cet1p homodimerization and Cet1p binding to the guanylyltransferase component of the capping apparatus. The structure and mechanism of fungal RNA triphosphatases are completely different from those of mammalian mRNA capping enzymes. Hence, RNA triphosphatase presents an ideal target for structure-based antifungal drug discovery.

Mutational analysis of 39 residues of vaccinia DNA topoisomerase identifies Lys-220, Arg-223, and Asn-228 as important for covalent catalysis.

Vaccinia DNA topoisomerase, a 314-amino acid type I enzyme, catalyzes the cleavage and rejoining of DNA strands through a DNA-(3′-phosphotyrosyl)-enzyme intermediate. To identify amino acids that participate in the transesterification reaction, we introduced alanine substitutions at 39 positions within a conserved 57amino acid segment upstream of the active-site tyrosine. Purified wild type and mutant proteins were compared with respect to their activities in relaxing supercoiled DNA. The majority of mutant proteins displayed wild type topoisomerase activity. Mutant enzymes that relaxed DNA at reduced rates were subjected to kinetic analysis of the strand cleavage and religation steps under single-turnover and equilibrium conditions. For the wild type topoisomerase, the observed single-turnover cleavage rate constant (kcl) was 0.29 s−1 and the cleavage-religation equilibrium constant (Kcl) was 0.22. The most dramatic mutational effects were seen with R223A; removal of the basic side chain reduced the rates of cleavage and religation by factors of 10−4.3 and 10−5.0, respectively, and shifted the cleavage-religation equilibrium in favor of the covalently bound state (Kcl = 1). Introduction of lysine at position 223 restored the rate of cleavage to 1/10 that of the wild type enzyme. We conclude that a basic residue is essential for covalent catalysis and suggest that Arg-223 is a constituent of the active site. Modest mutational effects were observed at two other positions (Lys-220 and Asn-228), at which alanine substitutions slowed the rates of strand cleavage by 1 order of magnitude and shifted the equilibrium toward the noncovalently bound state. Arg-223 and Lys-220 are conserved in all members of the eukaryotic type I topoisomerase family; Asn-228 is conserved among the poxvirus enzymes.

Structure–function analysis of the kinase-CPD domain of yeast tRNA ligase (Trl1) and requirements for complementation of tRNA splicing by a plant Trl1 homolog

Trl1 is an essential 827 amino acid enzyme that executes the end-healing and end-sealing steps of tRNA splicing in Saccharomyces cerevisiae. Trl1 consists of two domains—an N-terminal ligase component and a C-terminal 5′-kinase/2′,3′-cyclic phosphodiesterase (CPD) component—that can function in tRNA splicing in vivo when expressed as separate polypeptides. To understand the structural requirements for the kinase-CPD domain, we performed an alanine scan of 30 amino acids that are conserved in Trl1 homologs from other fungi. We thereby identified four residues (Arg463, His515, Thr675 and Glu741) as essential for activity in vivo. Structure–function relationships at these positions, and at four essential or conditionally essential residues defined previously (Asp425, Arg511, His673 and His777), were clarified by introducing conservative substitutions. Biochemical analysis showed that lethal mutations of Asp425, Arg463, Arg511 and His515 in the kinase module abolished polynucleotide kinase activity in vitro. We report that a recently cloned 1104 amino acid Arabidopsis RNA ligase functions in lieu of yeast Trl1 in vivo and identify essential side chains in the ligase, kinase and CPD modules of the plant enzyme. The plant ligase, like yeast Trl1 but unlike T4 RNA ligase 1, requires a 2′-PO4 end for tRNA splicing in vivo.

Essential constituents of the 3'-phosphoesterase domain of bacterial DNA ligase D, a nonhomologous end-joining enzyme.

DNA ligase D (LigD) catalyzes end-healing and end-sealing steps during nonhomologous end joining in bacteria. Pseudomonas aeruginosa LigD consists of a central ATP-dependent ligase domain fused to a C-terminal polymerase domain and an N-terminal 3′-phosphoesterase (PE) module. The PE domain catalyzes manganese-dependent phosphodiesterase and phosphomonoesterase reactions at a duplex primer-template with a short 3′-ribonucleotide tract. The phosphodiesterase, which cleaves a 3′-terminal diribonucleotide to yield a primer strand with a ribonucleoside 3′-PO4 terminus, requires the vicinal 2′-OH of the penultimate ribose. The phosphomonoesterase converts the terminal ribonucleoside 3′-PO4 to a 3′-OH. Here we show that the PE domain has a 3′-phosphatase activity on an all-DNA primer-template, signifying that the phosphomonoesterase reaction does not depend on a 2′-OH. The distinctions between the phosphodiesterase and phosphomonoesterase activities are underscored by the results of alanine-scanning, limited proteolysis, and deletion analysis, which show that the two reactions depend on overlapping but nonidentical ensembles of protein functional groups, including: (i) side chains essential for both ribonuclease and phosphatase activity (His-42, His-48, Asp-50, Arg-52, His-84, and Tyr-88); (ii) side chains important for 3′-phosphatase activity but not for 3′ ribonucleoside removal (Arg-14, Asp-15, Glu-21, Gln-40, and Glu-82); and (iii) side chains required selectively for the 3′-ribonuclease (Lys-66 and Arg-76). These constellations of critical residues are unique to LigD-like proteins, which we propose comprise a new bifunctional phosphoesterase family.

The adenylyltransferase domain of bacterial Pnkp defines a unique RNA ligase family

Pnkp is the end-healing and end-sealing component of an RNA repair system present in diverse bacteria from ten different phyla. To gain insight to the mechanism and evolution of this repair system, we determined the crystal structures of the ligase domain of Clostridium thermocellum Pnkp in three functional states along the reaction pathway: apoenzyme, ligase•ATP substrate complex, and covalent ligase-AMP intermediate. The tertiary structure is composed of a classical ligase nucleotidyltransferase module that is embellished by a unique α-helical insert module and a unique C-terminal α-helical module. Structure-guided mutational analysis identified active site residues essential for ligase adenylylation. Pnkp defines a new RNA ligase family with signature structural and functional properties.

Structures of bacterial polynucleotide kinase in a Michaelis complex with GTP•Mg2+ and 5′-OH oligonucleotide and a product complex with GDP•Mg2+ and 5′-PO4 oligonucleotide reveal a mechanism of general acid-base catalysis and the determinants of phosphoacceptor recognition

Clostridium thermocellum polynucleotide kinase (CthPnk), the 5′ end-healing module of a bacterial RNA repair system, catalyzes reversible phosphoryl transfer from an NTP donor to a 5′-OH polynucleotide acceptor. Here we report the crystal structures of CthPnk-D38N in a Michaelis complex with GTP•Mg2+ and a 5′-OH oligonucleotide and a product complex with GDP•Mg2+ and a 5′-PO4 oligonucleotide. The O5′ nucleophile is situated 3.0 Å from the GTP γ phosphorus in the Michaelis complex, where it is coordinated by Asn38 and is apical to the bridging β phosphate oxygen of the GDP leaving group. In the product complex, the transferred phosphate has undergone stereochemical inversion and Asn38 coordinates the 5′-bridging phosphate oxygen of the oligonucleotide. The D38N enzyme is poised for catalysis, but cannot execute because it lacks Asp38—hereby implicated as the essential general base catalyst that abstracts a proton from the 5′-OH during the kinase reaction. Asp38 serves as a general acid catalyst during the ‘reverse kinase’ reaction by donating a proton to the O5′ leaving group of the 5′-PO4 strand. The acceptor strand binding mode of CthPnk is distinct from that of bacteriophage T4 Pnk.

Structure-guided Mutational Analysis of the OB, HhH, and BRCT Domains of Escherichia coli DNA Ligase

NAD+-dependent DNA ligases (LigAs) are ubiquitous in bacteria and essential for growth. LigA enzymes have a modular structure in which a central catalytic core composed of nucleotidyltransferase and oligonucleotide-binding (OB) domains is linked via a tetracysteine zinc finger to distal helix-hairpin-helix (HhH) and BRCT (BRCA1-like C-terminal) domains. The OB and HhH domains contribute prominently to the protein clamp formed by LigA around nicked duplex DNA. Here we conducted a structure-function analysis of the OB and HhH domains of Escherichia coli LigA by alanine scanning and conservative substitutions, entailing 43 mutations at 22 amino acids. We thereby identified essential functional groups in the OB domain that engage the DNA phosphodiester backbone flanking the nick (Arg333); penetrate the minor grove and distort the nick (Val383 and Ile384); or stabilize the OB fold (Arg379). The essential constituents of the HhH domain include: four glycines (Gly455, Gly489, Gly521, Gly553), which bind the phosphate backbone across the minor groove at the outer margins of the LigA-DNA interface; Arg487, which penetrates the minor groove at the outer margin on the 3 ®-OH side of the nick; and Arg446, which promotes protein clamp formation via contacts to the nucleotidyltransferase domain. We find that the BRCT domain is required in its entirety for effective nick sealing and AMP-dependent supercoil relaxation.

Structure and mechanism of the 2′,3′ phosphatase component of the bacterial Pnkp-Hen1 RNA repair system

Pnkp is the end-healing and end-sealing component of an RNA repair system present in diverse bacteria from many phyla. Pnkp is composed of three catalytic modules: an N-terminal polynucleotide 5′ kinase, a central 2′,3′ phosphatase and a C-terminal ligase. The phosphatase module is a Mn2+-dependent phosphodiesterase–monoesterase that dephosphorylates 2′,3′-cyclic phosphate RNA ends. Here we report the crystal structure of the phosphatase domain of Clostridium thermocellum Pnkp with Mn2+ and citrate in the active site. The protein consists of a core binuclear metallo-phosphoesterase fold (exemplified by bacteriophage λ phosphatase) embellished by distinctive secondary structure elements. The active site contains a single Mn2+ in an octahedral coordination complex with Asp187, His189, Asp233, two citrate oxygens and a water. The citrate fills the binding site for the scissile phosphate, wherein it is coordinated by Arg237, Asn263 and His264. The citrate invades the site normally occupied by a second metal (engaged by Asp233, Asn263, His323 and His376), and thereby dislocates His376. A continuous tract of positive surface potential flanking the active site suggests an RNA binding site. The structure illuminates a large body of mutational data regarding the metal and substrate specificity of Clostridium thermocellum Pnkp phosphatase.