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1.  Crystal Structure of the Human NKX2.5 Homeodomain in Complex with DNA Target 
Biochemistry  2012;51(32):6312-6319.
NKX2.5 is a homeodomain containing transcription factor regulating cardiac formation and function, and its mutations are linked to congenital heart disease. Here we provide the first report of the crystal structure of the NKX2.5 homeodomain in complex with double-stranded DNA of its endogenous target, locating within the proximal promoter –242 site of the atrial natriuretic factor gene. The crystal structure, determined at 1.8 Å resolution, demonstrates that NKX2.5 homeodomains occupy both DNA binding sites separated by five nucleotides without physical interaction between themselves. The two homeodomains show identical conformation despite the differences in the DNA sequences they bind, and no significant bending of the DNA was observed. Tyr54, absolutely conserved in NK2 family proteins, mediates sequence-specific interaction with the TAAG motif. This high resolution crystal structure of NKX2.5 protein provides a detailed picture of protein and DNA interactions, which allows us to predict DNA binding of mutants identified in human patients.
doi:10.1021/bi300849c
PMCID: PMC3448007  PMID: 22849347
2.  Synchrotron Radiation Provides a Plausible Explanation for the Generation of a Free Radical Adduct of Thioxolone in Mutant Carbonic Anhydrase II 
Thioxolone acts as a prodrug in the presence of carbonic anhydrase II (CA II), whereby the molecule is cleaved by thioester hydrolysis to the carbonic anhydrase inhibitor, 4-mercaptobenzene-1,3-diol (TH0). Thioxolone was soaked into the proton transfer mutant H64A of CA II in an effort to capture a reaction intermediate via X-ray crystallography. Structure determination of the 1.2 Å resolution data revealed the TH0 had been modified to a 4,4′-disulfanediyldibenzene-1,3-diol, a product of crystallization conditions, and a zinc ligated 2,4-dihydroxybenzenesulfenic acid, most likely induced by radiation damage. Neither ligand was likely a result of an enzymatic mechanism.
doi:10.1021/jz100954h
PMCID: PMC2957018  PMID: 20976122
Carbonic anhydrase; free radical damage; synchrotron radiation; thioxolone; sulfenic acid
3.  High-resolution structure of human carbonic anhydrase II complexed with acetazolamide reveals insights into inhibitor drug design 
The crystal structure of human carbonic anhydrase II (CA II) complexed with acetazolamide (AZM) has been determined at 1.1 Å resolution. The co-binding of AZM and glycerol in the active site demonstrate that an isozyme specific CA inhibitor may be developed.
The crystal structure of human carbonic anhydrase II (CA II) complexed with the inhibitor acetazolamide (AZM) has been determined at 1.1 Å resolution and refined to an R cryst of 11.2% and an R free of 14.7%. As observed in previous CA II–inhibitor complexes, AZM binds directly to the zinc and makes several key interactions with active-site residues. The high-resolution data also showed a glycerol molecule adjacent to the AZM in the active site and two additional AZMs that are adventitiously bound on the surface of the enzyme. The co-binding of AZM and glycerol in the active site demonstrate that given an appropriate ring orientation and substituents, an isozyme-specific CA inhibitor may be developed.
doi:10.1107/S1744309109036665
PMCID: PMC2765883  PMID: 19851004
human carbonic anhydrase II; acetazolamide; inhibitor design
4.  Design of a Carbonic Anhydrase IX Active-Site Mimic to Screen Inhibitors for Possible Anti-Cancer Properties†,‡ 
Biochemistry  2009;48(6):1322-1331.
Recently a convincing body of evidence has accumulated, suggesting that the over-expression of carbonic anhydrase isozyme IX (CA IX) in some cancers contributes to the acidification of the extracellular matrix, which in turn promotes the growth and metastasis of the tumor. These observations have made CA IX an attractive drug target for the selective treatment of certain cancers. Currently there is no available x-ray crystal structure of CA IX and this has hampered the rational design of selective CA IX inhibitors. In light of these observations and based on structural alignment homology, using the crystal structure CA II and the sequence of CA IX, a double mutant of CA II with Ala 65 replaced by Ser and Asn 67 replace by Gln has been constructed to resemble the active site of CA IX. This CA IX mimic has been characterized kinetically using 18O-exchange and structurally using x-ray crystallography, alone and in complex with five CA sulfonamide based inhibitors; acetazolamide, benzolamide, chlorzolamide, ethoxzolamide, and methazolamide, and compared to CA II. This structural information has been evaluated in relationship to inhibition studies and in vitro cytotoxicity assays and shows a correlated structure-activity relationship. Kinetic and structural studies of CA II and CA IX mimic reveal chlorzolamide to be a more potent inhibitor of CA IX inducing an active site conformational change upon binding. Additionally, chlorzolamide appears to be cytotoxic to prostate cancer cells. This preliminary study demonstrates that the CA IX mimic may provide a useful model to design more isozyme specific CA IX inhibitors which may lead to development of new therapeutic treatments of some cancers.
doi:10.1021/bi802035f
PMCID: PMC2713499  PMID: 19170619
5.  Crystallization and preliminary X-ray analysis of the NKX2.5 homeodomain in complex with DNA 
The NKX2.5 homeodomain has been crystallized in complex with DNA. Diffraction data were collected to 1.7 Å resolution.
As part of an effort to elucidate the molecular basis for the pathogenesis of NKX2.5 mutations in congenital heart disease using X-ray crystallography, the NKX2.5 homeodomain has been crystallized in complex with a specific DNA element, the −242 promoter region of atrial natriuretic factor. Crystals of the homeodomain–DNA complex diffracted X-rays to 1.7 Å resolution and belonged to space group P65, with unit-cell parameters a = b = 71.5, c = 94.3 Å. The asymmetric unit contained two molecules of the NKX2.5 homeodomain and one double-stranded oligonucleotide.
doi:10.1107/S1744309108033447
PMCID: PMC2581709  PMID: 18997347
NKX2.5 homeodomain; congenital heart disease
6.  Crystallization and preliminary X-ray analysis of the NKX2.5 homeodomain in complex with DNA 
As part of an effort to elucidate the molecular basis for the pathogenesis of NKX2.5 mutations in congenital heart disease using X-ray crystallography, the NKX2.5 homeodomain has been crystallized in complex with a specific DNA element, the −242 promoter region of atrial natriuretic factor. Crystals of the homeodomain–DNA complex diffracted X-rays to 1.7 Å resolution and belonged to space group P65, with unit-cell parameters a = b = 71.5, c = 94.3 Å. The asymmetric unit contained two molecules of the NKX2.5 homeodomain and one double-stranded oligonucleotide.
doi:10.1107/S1744309108033447
PMCID: PMC2581709  PMID: 18997347

Results 1-6 (6)