Structural studies of glyceraldehyde-3-phosphate dehydrogenase complexes and the E. coli PutA DNA binding domain

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file.Title from title screen of research.pdf file (viewed on April 27, 2009)Vita.Includes bibliographical references.Thesis (Ph.D.) University of Missouri-Columbia 2006.Dissertations, Academic -- University of Missouri--Columbia -- Biochemistry (Agriculture)Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that catalyzes the formation of 1,3-bisphosphoglycerate from glyceraldehyde-3-phosphate. We have solved a high-resolution (1.75 Å) structure of a human GAPDH. Human GAPDH and the E3 ubiquitin ligase Siah1 have been found to interact as part of recently discovered NO/GAPDH/Siah1 apoptosis cascade. The structure is used in a computational ligand-docking study of the small-molecule compound CGP-3466, which inhibits apoptosis by preventing GAPDH binding to Siah1. The structure is also used to build a qualitative model of the complex between GAPDH-Siah1. We have also solved three crystal structures of Thermus aquaticus GAPDH corresponding to phosphate concentrations of 0 (1.65 Å), 50 mM (1.85 Å), and 100 mM (2.23 Å). In these structures the binding of phosphate results in two conformations of the phosphate binding loop and the dual occupancy of both the new and classical Pi-sites. Finally, Proline utilization A (PutA) is a membrane-associated bi-functional enzyme that catalyzes the sequential two-step oxidation of proline to glutamate. Here we report the first crystal structure of a PutA DNA-binding domain along with functional analysis of a mutant PutA defective in DNA-binding

U2AF65 adapts to diverse pre-mRNA splice sites through conformational selection of specific and promiscuous RNA recognition motifs

Degenerate splice site sequences mark the intron boundaries of pre-mRNA transcripts in multicellular eukaryotes. The essential pre-mRNA splicing factor U2AF(65) is faced with the paradoxical tasks of accurately targeting polypyrimidine (Py) tracts preceding 3\u27 splice sites while adapting to both cytidine and uridine nucleotides with nearly equivalent frequencies. To understand how U2AF(65) recognizes degenerate Py tracts, we determined six crystal structures of human U2AF(65) bound to cytidine-containing Py tracts. As deoxy-ribose backbones were required for co-crystallization with these Py tracts, we also determined two baseline structures of U2AF(65) bound to the deoxy-uridine counterparts and compared the original, RNA-bound structure. Local structural changes suggest that the N-terminal RNA recognition motif 1 (RRM1) is more promiscuous for cytosine-containing Py tracts than the C-terminal RRM2. These structural differences between the RRMs were reinforced by the specificities of wild-type and site-directed mutant U2AF(65) for region-dependent cytosine- and uracil-containing RNA sites. Small-angle X-ray scattering analyses further demonstrated that Py tract variations select distinct inter-RRM spacings from a pre-existing ensemble of U2AF(65) conformations. Our results highlight both local and global conformational selection as a means for universal 3\u27 splice site recognition by U2AF(65)

A Broad Range of Conformations Contribute to the Solution Ensemble of the Essential Splicing Factor U2AF⁶⁵

U2AF⁶⁵ is essential for pre-mRNA splicing in most eukaryotes. Two consecutive RNA recognition motifs (RRM) of U2AF⁶⁵ recognize a polypyrimidine tract at the 3′ splice site. Here, we use small-angle X-ray scattering to demonstrate that the tandem U2AF⁶⁵ RRMs exhibit a broad range of conformations in the solution ensemble. The majority of U2AF⁶⁵ conformations exhibit few contacts between the RRMs, such as observed in the crystal structure. A subpopulation adopts tight inter-RRM contacts, such as independently reported based on paramagnetic relaxation enhancements. These complementary structural methods demonstrate that diverse splice sites have the opportunity to select compact or extended inter-RRM proximities from the U2AF⁶⁵ conformational pool

Cancer-Associated Mutations Mapped on High-Resolution Structures of the U2AF2 RNA Recognition Motifs

Acquired point mutations of pre-mRNA splicing factors recur among cancers, leukemias, and related neoplasms. Several studies have established that somatic mutations of a U2AF1 subunit, which normally recognizes 3′ splice site junctions, recur among myelodysplastic syndromes. The U2AF2 splicing factor recognizes polypyrimidine signals that precede most 3′ splice sites as a heterodimer with U2AF1. In contrast with those of the well-studied U2AF1 subunit, descriptions of cancer-relevant U2AF2 mutations and their structural relationships are lacking. Here, we survey databases of cancer-associated mutations and identify recurring missense mutations in the U2AF2 gene. We determine ultra-high-resolution structures of the U2AF2 RNA recognition motifs (RRM1 and RRM2) at 1.1 Å resolution and map the structural locations of the mutated U2AF2 residues. Comparison with prior, lower-resolution structures of the tandem U2AF2 RRMs in the RNA-bound and apo states reveals clusters of cancer-associated mutations at the U2AF2 RRM–RNA or apo-RRM1–RRM2 interfaces. Although the role of U2AF2 mutations in malignant transformation remains uncertain, our results show that cancer-associated mutations correlate with functionally important surfaces of the U2AF2 splicing factor

Structural Basis of the Transcriptional Regulation of the Proline Utilization Regulon by Multifunctional PutA

The multifunctional Escherichia coli proline utilization A (PutA) flavoprotein functions both as a membrane-associated proline catabolic enzyme and as a transcriptional repressor of the proline utilization genes putA and putP. To better understand the mechanism of transcriptional regulation by PutA, we have mapped the put-regulatory region, determined a crystal structure of the PutA ribbon–helix–helix domain (PutA52, a polypeptide corresponding to residues 1–52 of E. coli PutA) complexed with DNA, and examined the thermodynamics of DNA binding to PutA52. Five operator sites, each containing the sequence motif 5′-GTTGCA-3′, were identified using gelshift analysis. Three of the sites are shown to be critical for repression of putA, whereas the two other sites are important for repression of putP. The 2.25-Å-resolution crystal structure of PutA52 bound to one of the operators (operator 2; 21 bp) shows that the protein contacts a 9-bp fragment corresponding to the GTTGCA consensus motif plus three flanking base pairs. Since the operator sequences differ in flanking bases, the structure implies that PutA may have different affinities for the five operators. This hypothesis was explored using isothermal titration calorimetry. The binding of PutA52 to operator 2 is exothermic, with an enthalpy of −1.8 kcal/mol and a dissociation constant of 210 nM. Substitution of the flanking bases of operator 4 into operator 2 results in an unfavorable enthalpy of 0.2 kcal/mol and a 15-fold-lower affinity, showing that base pairs outside of the consensus motif impact binding. Structural and thermodynamic data suggest that hydrogen bonds between Lys9 and bases adjacent to the GTTGCA motif contribute to transcriptional regulation by fine-tuning the affinity of PutA for put control operators