Katherine A. Kantardjieff

Matthews coefficient probabilities: Improved estimates for unit cell contents of proteins, DNA, and protein–nucleic acid complex crystals

Estimating the number of molecules in the crystallographic asymmetric unit is one of the first steps in a macromolecular structure determination. Based on a survey of 15,641 crystallographic Protein Data Bank (PDB) entries the distribution of VM, the crystal volume per unit of protein molecular weight, known as Matthews coefficient, has been reanalyzed. The range of values and frequencies has changed in the 30 years since Matthews first analysis of protein crystal solvent content. In the statistical analysis, complexes of proteins and nucleic acids have been treated as a separate group. In addition, the VM distribution for nucleic acid crystals has been examined for the first time. Observing that resolution is a significant discriminator of VM, an improved estimator for the probabilities of the number of molecules in the crystallographic asymmetric unit has been implemented, using resolution as additional information.

Protein isoelectric point as a predictor for increased crystallization screening efficiency

MOTIVATION Increased efficiency in initial crystallization screening reduces cost and material requirements in structural genomics. Because pH is one of the few consistently reported parameters in the Protein Data Bank (PDB), the isoelectric point (pI) of a protein has been explored as a useful indirect predictor for the optimal choice of range and distribution of the pH sampling in crystallization trials. RESULTS We have analyzed 9596 unique protein crystal forms from the August 2003 PDB and have found a significant relationship between the calculated pI of successfully crystallized proteins and the difference between pI and reported pH at which they were crystallized. These preferences provide strong prior information for the design of crystallization screening experiments with significantly increased efficiency and corresponding reduction in material requirements, leading to potential cost savings of millions of US

Concanavalin A in a dimeric crystal form: revisiting structural accuracy and molecular flexibility

for structural genomics projects involving high-throughput crystallographic structure determination. AVAILABILITY A prototype example of a screen design and efficiency estimator program, CrysPred, is available at http://www-structure.llnl.gov/cryspred/

Structure of GES-1 at Atomic Resolution: Insights Into the Evolution of Carbapenamase Activity in the Class a Extended-Spectrum Beta-Lactamases

A structure of native concanavalin A (ConA), a hardy perennial of structural biology, has been determined in a dimeric crystal form at a resolution of 1.56 A (space group C222(1); unit-cell parameters a = 118.70, b = 101.38, c = 111.97 A; two molecules in the asymmetric unit). The structure has been refined to an R(free) of 0.206 (R = 0.178) after iterative model building and phase-bias removal using Shake&wARP. Correspondence between calculated water-tyrosine interactions and experimentally observed structures near the saccharide-binding site suggests that the observed interactions between Tyr12 and water in various crystal forms are to be expected and are not unique to the presence of an active site. The present structure differs from previously reported atomic resolution structures of ConA in several regions and extends insight into the conformational flexibility of this molecule. Furthermore, this third, low-temperature, structure of ConA in a different crystal form, independently refined using powerful model-bias removal techniques, affords the opportunity to revisit assessment of accuracy and precision in high- or atomic resolution protein structures. It is illustrated that several precise structures of the same molecule can differ substantially in local detail and users of crystallographic models are reminded to consider the potential impact when interpreting structures. Suggestions on how to effectively represent ensembles of crystallographic models of a given molecule are provided.

Mycobacterium tuberculosis RmlC epimerase (Rv3465): a promising drug‐target structure in the rhamnose pathway

The structure of the class A extended-spectrum beta-lactamase GES-1 from Klebsiella pneumoniae has been determined to 1.1 A resolution. GES-1 has the characteristic active-site disulfide bond of the carbapenemase family of beta-lactamases and has a structure that is very similar to those of other known carbapenemases, including NMC-A, SME-1 and KPC-2. Most residues implicated in the catalytic mechanism of this class of enzyme are present in the GES-1 active site, including Ser70, which forms a covalent bond with the carbonyl C atom of the beta-lactam ring of the substrate during the formation of an acyl-enzyme intermediate, Glu166, which is implicated as both the acylation and deacylation base, and Lys73, which is also implicated as the acylation base. A water molecule crucial to catalysis is observed in an identical location as in other class A beta-lactamases, interacting with the side chains of Ser70 and Glu166. One important residue, Asn170, also normally a ligand for the hydrolytic water, is missing from the GES-1 active site. This residue is a glycine in GES-1 and the enzyme is unable to hydrolyze imipenem. This points to this residue as being critically important in the hydrolysis of this class of beta-lactam substrate. This is further supported by flexible-docking studies of imipenem with in silico-generated Gly170Asn and Gly170Ser mutant GES-1 enzymes designed to mimic the active sites of imipenem-hydrolyzing point mutants GES-2 and GES-5.

Laboratory scale structural genomics

The Mycobacterium tuberculosis rmlC gene encodes dTDP-4-keto-6-deoxyglucose epimerase, the third enzyme in the M. tuberculosis dTDP-L-rhamnose pathway which is essential for mycobacterial cell-wall synthesis. Because it is structurally unique, highly substrate-specific and does not require a cofactor, RmlC is considered to be the most promising drug target in the pathway, and the M. tuberculosis rmlC gene was selected in the initial round of TB Structural Genomics Consortium targets for structure determination. The 1.7 A native structure determined by the consortium facilities is reported and implications for in silico screening of ligands for structure-guided drug design are discussed.

Structural bioinformatic approaches to the discovery of new antimycobacterial drugs.

At Lawrence Livermore National Laboratory, the development of the TB structural genomics consortium crystallization facility has paralleled several local proteomics research efforts that have grown out of gene expression microarray and comparative genomics studies. Collective experience gathered from TB consortium labs and other centers involved in the NIH-NIGMS protein structure initiative allows us to explore the possibilities and challenges of pursuing structural genomics on an academic laboratory scale. We discuss our procedures and protocols for genomic targeting approaches, primer design, cloning, small scale expression screening, scale-up and purification, through to automated crystallization screening and data collection. The procedures are carried out by a small group using a combination of traditional approaches, innovative molecular biochemistry approaches, software automation, and a modest investment in robotic equipment.

Highly enantioselective mutant carbonyl reductases created via structure-based site-saturation mutagenesis.

Integrated bioinformatic approaches to drug discovery exploit computational techniques to examine the flow of information from genome to structure to function. Informatics is being be used to accelerate and rationalize the process of antimycobacterial drug discovery and design, with the immediate goals to identify viable drug targets and produce a set of critically evaluated protein target models and corresponding set of probable lead compounds. Bioinformatic approaches are being successfully applied in the selection and prioritization of putative mycobacterial drug target genes; computational modelling and x-ray structure validation of protein targets with drug lead compounds; simulated docking and virtual screening of potential lead compounds; and lead validation and optimization using structure-activity and structure-function relationships. By identifying active sites, characterizing patterns of conserved residues and, where relevant, predicting catalytic residues, bioinformatics provides information to aid the design of selective and efficacious pharmacophores. In this review, we describe selected recent progress in antimycobacterial drug design, illustrating the strengths and limitations of current structural bioinformatic approaches as tools in the fight against tuberculosis.

Structure of pyrR (Rv1379) from Mycobacterium tuberculosis: a persistence gene and protein drug target

A carbonyl reductase from Sporobolomyces salmonicolor reduced para-substituted acetophenones with low enantioselectivity. Enzyme-substrate docking studies revealed that residues M242 and Q245 were in close proximity to the para-substituent of acetophenones in the substrate binding site. Site-saturation mutagenesis of M242 or Q245, and double mutation of M242 and Q245 were performed in order to enhance the enzymes enantioselectivity toward the reduction of para-substituted acetophenones. Three Q245 mutants were obtained, which inverted the enantiopreference of product alcohols from (R)- to (S)-configuration with high ee values (Org. Lett. 2008, 10, 525-528). Four M242 mutant enzymes also showed greater preference for the formation of (S)-enantiomeric alcohols than the wild-type enzyme, but to a much less extent than Q245 mutants. M242/Q245 double variations not only greatly affect the enantiomeric purity of the product alcohols, but also invert the enantiopreference, demonstrating that these residues play a critical role in determining the enantioselectivity of these ketone reductions. The kinetic parameters of these mutant enzymes indicated that residues 242 and 245 also exert an effect on the catalytic activity of this carbonyl reductase. Highly enantioselective mutant carbonyl reductases were created by site-saturation mutagenesis, among which the one bearing double mutations, M242L/Q245P, showed the highest enantioselectivity that catalyzed the reduction of the tested para-substituted acetophenones to give (S)-enantiomeric products in ≥99% ee with only one exception of p-fluoroacetophenone (92% ee).

The solvent component of macromolecular crystals

The Mycobacterium tuberculosis pyrR gene (Rv1379) encodes a protein that regulates the expression of pyrimidine-nucleotide biosynthesis (pyr) genes in a UMP-dependent manner. Because pyrimidine biosynthesis is an essential step in the progression of TB, the gene product pyrR is an attractive antitubercular drug target. The 1.9 A native structure of Mtb pyrR determined by the TB Structural Genomics Consortium facilities in trigonal space group P3(1)21 is reported, with unit-cell parameters a = 66.64, c = 154.72 A at 120 K and two molecules in the asymmetric unit. The three-dimensional structure and residual uracil phosphoribosyltransferase activity point to a common PRTase ancestor for pyrR. However, while PRPP- and UMP-binding sites have been retained in Mtb pyrR, a distinct dimer interaction among subunits creates a deep positively charged cleft capable of binding pyr mRNA. In silico screening of pyrimidine-nucleoside analogs has revealed a number of potential lead compounds that, if bound to Mtb pyrR, could facilitate transcriptional attenuation, particularly cyclopentenyl nucleosides.