Todd Weaver

Missense Mutations in Fumarate Hydratase in Multiple Cutaneous and Uterine Leiomyomatosis and Renal Cell Cancer

Heterozygous germline mutations in fumarate hydratase (FH) predispose to the multiple cutaneous and uterine leiomyomatosis syndrome (MCUL), which, when co-existing with renal cancer, is also known as hereditary leiomyomatosis and renal cell cancer. Twenty-seven distinct missense mutations represent 68% of FH mutations reported in MCUL. Here we show that FH missense mutations significantly occurred in fully conserved residues and in residues functioning in the FH A-site, B-site, or subunit-interacting region. Of 24 distinct missense mutations, 13 (54%) occurred in the substrate-binding A-site, 4 (17%) in the substrate-binding B-site, and 7 (29%) in the subunit-interacting region. Clustering of missense mutations suggested the presence of possible mutational hotspots. FH functional assay of lymphoblastoid cell lines from 23 individuals with heterozygous FH missense mutations showed that A-site mutants had significantly less residual activity than B-site mutants, supporting data from Escherichia coli that the A-site is the main catalytic site. Missense FH mutations predisposing to renal cancer had no unusual features, and identical mutations were found in families without renal cancer, suggesting a role for genetic or environmental factors in renal cancer development in MCUL. That all missense FH mutations associating with MCUL/hereditary leiomyomatosis and renal cell cancer showed diminished FH enzymatic activity suggests that the tumor suppressor role of fumarate hydratase may relate to its enzymatic function.

X‐ray crystallographic and kinetic correlation of a clinically observed human fumarase mutation

Fumarase catalyzes the reversible conversion of fumarate to S‐ malate during the operation of the ubiquitous Krebs cycle. Previous studies have shown that the active site includes side chains from three of the four subunits within the tetrameric enzyme. We used a clinically observed human mutation to narrow our search for potential catalytic groups within the fumarase active site. Offspring homozygous for the missense mutation, a G‐955–C transversion in the fumarase gene, results in the substitution of a glutamine at amino acid 319 for the normal glutamic acid. To more fully understand the implications of this mutation, a single‐step site‐directed mutagenesis method was used to generate the homologous substitution at position 315 within fumarase C from Escherichia coli. Subsequent kinetic and X‐ray crystal structure analyses show changes in the turnover number and the cocrystal structure with bound citrate.

Structural and functional studies of truncated hemolysin A from Proteus mirabilis.

In this study we analyzed the structure and function of a truncated form of hemolysin A (HpmA265) from Proteus mirabilis using a series of functional and structural studies. Hemolysin A belongs to the two-partner secretion pathway. The two-partner secretion pathway has been identified as the most common protein secretion pathway among Gram-negative bacteria. Currently, the mechanism of action for the two-partner hemolysin members is not fully understood. In this study, hemolysis experiments revealed a unidirectional, cooperative, biphasic activity profile after full-length, inactive hemolysin A was seeded with truncated hemolysin A. We also solved the first x-ray structure of a TpsA hemolysin. The truncated hemolysin A formed a right-handed parallel β-helix with three adjoining segments of anti-parallel β-sheet. A CXXC disulfide bond, four buried solvent molecules, and a carboxyamide ladder were all located at the third complete β-helix coil. Replacement of the CXXC motif led to decreased activity and stability according to hemolysis and CD studies. Furthermore, the crystal structure revealed a sterically compatible, dry dimeric interface formed via anti-parallel β-sheet interactions between neighboring β-helix monomers. Laser scanning confocal microscopy further supported the unidirectional interconversion of full-length hemolysin A. From these results, a model has been proposed, where cooperative, β-strand interactions between HpmA265 and neighboring full-length hemolysin A molecules, facilitated in part by the highly conserved CXXC pattern, account for the template-assisted hemolysis.

Organelle and translocatable forms of glyoxysomal malate dehydrogenase

Many organelle enzymes coded for by nuclear genes have N‐terminal sequences, which directs them into the organelle (precursor) and are removed upon import (mature). The experiments described below characterize the differences between the precursor and mature forms of watermelon glyoxysomal malate dehydrogenase. Using recombinant protein methods, the precursor (p‐gMDH) and mature (gMDH) forms were purified to homogeneity using Ni2+–NTA affinity chromatography. Gel filtration and dynamic light scattering have shown both gMDH and p‐gMDH to be dimers in solution with p‐gMDH having a correspondingly higher molecular weight. p‐gMDH also exhibited a smaller translational diffusion coefficient (Dt) at temperatures between 4 and 32 °C resulting from the extra amino acids on the N‐terminal. Differential scanning calorimetry described marked differences in the unfolding properties of the two proteins with p‐gMDH showing additional temperature dependent transitions. In addition, some differences were found in the steady state kinetic constants and the pH dependence of the Km for oxaloacetate. Both the organelle‐precursor and the mature form of this glyoxysomal enzyme were crystallized under identical conditions. The crystal structure of p‐gMDH, the first structure of a cleavable and translocatable protein, was solved to a resolution of 2.55 Å. GMDH is the first glyoxysomal MDH structure and was solved to a resolution of 2.50 Å. A comparison of the two structures shows that there are few visible tertiary or quaternary structural differences between corresponding elements of p‐gMDH, gMDH and other MDHs. Maps from both the mature and translocatable proteins lack significant electron density prior to G44. While no portion of the translocation sequences from either monomer in the biological dimer was visible, all of the other solution properties indicated measurable effects of the additional residues at the N‐terminal.

Exploring Protein Function and Evolution Using Free Online Bioinformatics Tools

Bioinformatics provides a set of powerful research tools for predicting the function of a newly discovered protein and has quickly become an important field of training at many universities and medical institutions [1, 2]. Bioinformatics can also be used to explore regions of similarity and identity within families of proteins (paralogs) and across species (orthologs). Because the number of protein families with available three-dimensional (3D) structures has increased 3-fold over the past 8 years, an integrative approach may be taken in which paralog and ortholog sequence alignments are superimposed and visualized onto a representative 3D structure [3]. The teaching philosophy within undergraduate laboratory courses has also shifted away from cookbook experiments toward guided inquiry. The field of bioinformatics is an excellent choice for a guided-inquiry experience because of the huge databases and wide variety of questions students can ask [4]. The following laboratory exercise highlights the successful integration of bioinformatics into the undergraduate laboratory setting. The procedures involve analysis of an amino acid sequence for motifs, domains, secondary structure, and paralog and ortholog alignments as well as mapping these alignments onto a representative protein structure. The exercise emphasizes laboratory skills deemed critical for success of students within their scientific and medical careers [5].

Sequential unfolding of the hemolysin two‐partner secretion domain from Proteus mirabilis

Protein secretion is a major contributor to Gram‐negative bacterial virulence. Type Vb or two‐partner secretion (TPS) pathways utilize a membrane bound β‐barrel B component (TpsB) to translocate large and predominantly virulent exoproteins (TpsA) through a nucleotide independent mechanism. We focused our studies on a truncated TpsA member termed hemolysin A (HpmA265), a structurally and functionally characterized TPS domain from Proteus mirabilis. Contrary to the expectation that the TPS domain of HpmA265 would denature in a single cooperative transition, we found that the unfolding follows a sequential model with three distinct transitions linking four states. The solvent inaccessible core of HpmA265 can be divided into two different regions. The C‐proximal region contains nonpolar residues and forms a prototypical hydrophobic core as found in globular proteins. The N‐proximal region of the solvent inaccessible core, however, contains polar residues. To understand the contributions of the hydrophobic and polar interiors to overall TPS domain stability, we conducted unfolding studies on HpmA265 and site‐specific mutants of HpmA265. By correlating the effect of individual site‐specific mutations with the sequential unfolding results we were able to divide the HpmA265 TPS domain into polar core, nonpolar core, and C‐terminal subdomains. Moreover, the unfolding studies provide quantitative evidence that the folding free energy for the polar core subdomain is more favorable than for the nonpolar core and C‐terminal subdomains. This study implicates the hydrogen bonds shared among these conserved internal residues as a primary means for stabilizing the N‐proximal polar core subdomain.

Crystallization of truncated hemolysin A from Proteus mirabilis

Proteus species are second only to Escherichia coli as the most common causative agent of Gram-negative bacteria-based urinary-tract infections and many harbor several virulence factors that provide inherent uropathogenicity. One virulence factor stems from a two-partner secretion pathway comprised of hemolysin A and hemolysin B; upon hemolysin B-dependent secretion, hemolysin A becomes activated. This system is distinct from the classic type I secretion pathway exemplified by the hemolysin system within Escherichia coli. In order to describe the mechanism by which hemolysin A is activated for pore formation, an amino-terminal truncated form capable of complementing the non-secreted full-length hemolysin A and thereby restoring hemolytic activity has been constructed, expressed and purified. A room-temperature data set has been collected to 2.5 A resolution. The crystal belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 34.47, b = 58.40, c = 119.74 A. The asymmetric unit is expected to contain a single monomer, which equates to a Matthews coefficient of 1.72 A3 Da(-1) and a solvent content of 28.3%.