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Summary: Florante Quiocho is interested in understanding the atomic details of protein-ligand interactions, processes that form the basis of biological and biochemical specificity and activity.

The precise interactions between proteins and their targets (which may include other proteins) are the basis of biological specificity and activity. Anchored by x-ray crystallographic analysis, our research is directed toward understanding a wide spectrum of these interactions at the atomic level.

	The VP5 subunit of the herpes simplex virus...

The Role of Conformational Changes of the ATPase Subunits of ABC Transporter
ABC (ATP-binding cassette) transport systems are a large class of transporters that function from bacteria to humans in coupling the energy of ATP hydrolysis to the translocation of substances across the cell membrane. Overexpression of human P-glycoprotein in tumor cells following chemotherapy can contribute to multidrug resistance, and several human diseases have been traced to ABC proteins, including cystic fibrosis, hyperinsulinemia, and macular dystrophy. The bacterial ABC maltose transport system is composed of a periplasmic primary ligand receptor (MalE), two transmembrane-spanning subunits (MalF and MalG), and two ATPase subunits (MalK₂) at the cytosolic membrane surface. MalK consists of an amino-terminal nucleotide-binding domain (NBD) and a carboxyl-terminal regulatory domain (RD).

In collaboration with Jue Chen (formerly an HHMI associate in this lab, now at Purdue University) and Amy Davidson (Baylor College of Medicine), we have determined three MalK₂ structures, one with and two without bound ATP. The three structures maintain similar extensive interactions between RDs, which contribute to the homodimer formation. In the ATP-bound structure, however, the NBDs are in close contact and bury two ATP molecules between them. In marked contrast, the two unbound structures show separation of the NBDs at two different distances, indicating a tweezers-like motion of the ATPase dimer. These results provide the first concrete evidence for a structural change of the ATPase dimer—through a hinge bending between the NBD and RD domains—that modulates ATP hydrolysis and gating for a unidirectional ligand translocation process in ABC transporters.

Homing Endonucleases: Three-Dimensional Structures and Reagents for DNA Repair
The sequence of the human genome will facilitate the identification of genetic mutations that cause diseases, and a central goal in the postgenomic era will be to develop tools to correct these errors. The repair of complex genomes requires having reagents that can locate a sequence from among several hundred megabases of nonspecific DNA. Homing endonucleases, found in all branches of living organisms, are a novel class of enzymes that are able to recognize and cleave single target DNA sequences within the complex genomes. They promote the mobility of their encoding genes by generating a double-strand break at cognate alleles that lack their genes. The repair of the break by gene conversion results in the propagation of the homing endonuclease gene throughout the population. Homing endonucleases occur as open reading frames within introns or exist as integral parts of self-splicing inteins, and splicing of homing endonuclease sequences at the RNA or protein level prevents these elements from being deleterious to their host organism. The most remarkable feature of homing endonucleases is that they make numerous base-specific contacts within unusually long recognition sequences of 14–40 bp. A long-term goal is to recruit the extreme DNA sequence specificity of homing endonucleases to perform novel functions within living cells that facilitate diagnosis and therapy of genetic disorders. Moreover, with the atomic structures of the homing endonucleases as a basis, they could be modified by site-directed mutagenesis to recognize different base pairs, thus enlarging the available reagents.

In collaboration with Frederick Gimble (Texas A&M University), we have been engaged in the determination of the crystal structures of homing endonucleases. We previously determined the structure of PI-SceI, the first for a homing endonuclease and a protein generated by protein splicing, with and without a bound 36-bp substrate.

Recently we have determined the crystal structure of the yeast I-SceI homing endonuclease. I-SceI is the current reagent-of-choice to study DNA repair pathways that operate in yeast, plant, insect, fish, and mammalian systems because of its ability to target double-strand breaks at defined loci without affecting cell viability by cleaving at ectopic sites. Gene targeting has also been facilitated using this enzyme. Like PI-SceI, I-SceI is a monomer and contains two signature LAGLIDADG sequence motifs that define two catalytic sites each for cleaving one DNA strand. I-SceI recognizes and cleaves an asymmetric 18-bp DNA sequence to generate 4-bp extensions with 3' overhangs.

The structure of I-SceI contains a bound 24-bp DNA duplex and three calcium atoms, noncompetent analogs of magnesium, to prevent DNA cleavage. The structure is composed of two similar pseudosymmetric domains (identified as amino-terminal or amino- and carboxyl-terminal or C domains), each with a LADLIDAGD motif in an a helix. Both LADLIDAGD helices, which contain the catalytic aspartic residues, are located at the interface between the two domains. One calcium metal is shared between the two aspartates, while the two other metals are distributed in each active site. Although no significant bend is observed in the DNA bound to I-SceI, the minor groove at the I-SceI active sites is significantly compressed. The compression of the minor groove, which appears to be a prerequisite for DNA cleavage in LADLIDADG endonucleases, brings the scissile phosphates into the proximity of the active sites. The DNA compression in I-SceI is clearly asymmetric: more of the cleavage site of the bottom strand is buried than that of the top strand. The structure indicates that the bottom strand of the DNA substrate is cleaved first by one active site and then, following a structural rearrangement of the second site, the top strand is cut.

High Specificity of Phosphate Transport: The Structure of the Primary Receptor for the ABC Phosphate Transporter in Mycobacterium tuberculosis
One of the clearest manifestations of the importance of phosphate as a nutrient is the evolution of extremely high specificity of phosphate transport in cells, into mitochondria, and across brush borders. To understand this specificity, we solved the structure of phosphate-bound PstS-1, the initial cell surface receptor for the ABC phosphate transporter of M. tuberculosis. Because PstS-1 is the most immunodominant species-specific antigen of M. typhimurium, the tertiary structures of complexes of PstS-1 with antibodies will be valuable for rational selection of peptide epitopes for development of improved diagnostic reagents and subunit vaccine. Moreover, due to the pivotal role of PstS-1 in the phosphate-uptake system of mycobacteria, the structure could serve as a target molecule for drug design to combat tuberculosis.

The PstS-1 structure is composed of two similar globular domains that are bisected by a deep cleft wherein the inorganic phosphate is bound and completely buried. The phosphate, which is completely desolvated, is held in place by 13 hydrogen bonds, of which 11 are formed with NH and OH dipolar donor groups. The further presence of two carboxylate side chains confer stringent specificity by serving as negatively charged hydrogen bond acceptors of the proton(s) of the phosphate. The ion-dipole interactions between the phosphate and polar groups also play a major role in compensating for the isolated negative charges of the ligand. Surprisingly, the electrostatic surface in and around the cleft is intensely negative. This demonstrates the power of ion-dipole interactions in anion binding and electrostatic balance. Additional functional features include both the flexible amino-terminal segment that tethers PstS-1 on the cell surface and the hinge between the two domains, which should facilitate snaring the phosphate in the medium.

Last updated June 06, 2003