No abstract

The molecular structures of two human transthyretin (hTTR, prealbumin) complexes, co-crystallized with thyroxine (3,5,3',5'-tetraiodo-L-thyronine; T(4)), and with 3',5'-dinitro-N-acetyl-LL-thyronine (DNNAT), were determined by X-ray diffraction methods. Crystals of both structures are orthorhombic, space group P2(1)2(1)2, and have two independent monomers in the asymmetric unit of the crystal lattice. These structures have been refined to 17.0% for 8-2.0 A resolution data for the T(4) complex (I), and to R = 18.4% for 8-2.2 A resolution data for the DNNAT structure (II). This report provides a detailed description of T(4) binding to wild-type hTTR at 2.0 A resolution, as well as DNNAT. In both structures, the two independent hormone-binding sites of the TTR tetramer are occupied by ligand. A 50% statistical disorder model was applied to account for the crystallographic twofold symmetry along the binding channel and the lack of such symmetry for the ligands. Results for the co-crystallized T(4) complex show that T(4) binds deep in the hormone-binding channel and displaces the bound water previously reported for T(4) soaked into a native transthyretin crystal [Blake & Oatley (1977). Nature (London), 268, 115-120]. DNNAT also binds deeper in the channel toward the tetramer center than T(4) with the nitro groups occupying the symmetrical innermost halogen pockets. The N-acetyl moiety does not form polar contacts with the protein side chains as it is oriented toward the center of the channel. The weak binding affinity of DNNAT results from the loss of hydrophobic interactions with the halogen binding pockets as observed in T(4) binding. These data suggest that the halogen-binding sites toward the tetramer center are of primary importance as they are occupied by analogues with weak affinity to TTR, and are therefore selected over the other halogen sites which contribute more strongly to the overall binding affinity.

Crystallization has proven to be the most significant bottleneck to high-throughput protein structure determination using diffraction methods. We have used the large-scale, systematically generated experimental results of the Northeast Structural Genomics Consortium to characterize the biophysical properties that control protein crystallization. Datamining of crystallization results combined with explicit folding studies lead to the conclusion that crystallization propensity is controlled primarily by the prevalence of well-ordered surface epitopes capable of mediating interprotein interactions and is not strongly influenced by overall thermodynamic stability. These analyses identify specific sequence features correlating with crystallization propensity that can be used to estimate the crystallization probability of a given construct. Analyses of entire predicted proteomes demonstrate substantial differences in the bulk amino acid sequence properties of human versus eubacterial proteins that reflect likely differences in their biophysical properties including crystallization propensity. Finally, our thermodynamic measurements enable critical evaluation of previous claims regarding correlations between protein stability and bulk sequence properties, which generally are not supported by our dataset. NIH Public Access Author ManuscriptNat Biotechnol. Author manuscript; available in PMC 2010 January 1. Published in final edited form as:Nat Biotechnol. 2009 January ; 27(1): 51-57. doi:10.1038/nbt.1514. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe ability to determine the atomic structures of macromolecules represents a great achievement in molecular biology because of the unparalleled value of this information in understanding the fundamental chemistry of life [1][2][3][4][5] . While nuclear magnetic resonance represents an invaluable source of structural information, especially for small proteins, most macromolecular structures are determined using x-ray crystallography. Capitalizing on the recent proliferation of genomic sequence data, "structural genomics" consortia have been organized worldwide to develop methods and infrastructure for high-throughput protein structure determination. These groups have contributed to improvements in expression and structure determination methods 6 , and the four largest U.S. consortia accounted for 45% of all novel structures deposited in the Protein Data Bank (PDB) in 2007 7 . While these efforts contribute to the impressive progress of the structural biology community in characterizing the full repertoire of protein structures, the rate of growth in sequence information nonetheless far out-paces that of structural information. Given the ongoing acceleration of whole-genome sequencing, the gap between the two will continue to expand without a breakthrough in macromolecular structure determination methods.The systematic efforts of structural genomics projects show that crystallization is the major bottleneck to protein structure determinati...

The coleopteran-active delta-endotoxin Cry3Bb1 from Bacillus thuringiensis (Bt) strain EG7231 is uniquely toxic to Diabrotica undecimpunctata, the Southern corn rootworm, while retaining activity against Leptinotarsa decemlineata, the Colorado potato beetle. The crystal structure of the delta-endotoxin Cry3Bb1 has been refined using data collected to 2.4 A resolution, with a residual R factor of 17.5% and an R(free) of 25.3%. The structure is made up of three domains: I, a seven-helix bundle (residues 64-294); II, a three-sheet domain (residues 295-502); and III, a beta-sandwich domain (residues 503-652). The monomers in the orthorhombic C222(1) crystal lattice form a dimeric quaternary structure across a crystallographic twofold axis, with a channel formed involving interactions between domains I and III. There are 23 hydrogen bonds between the two monomers conferring structural stability on the dimer. It has been demonstrated that Cry3Bb1 and the similar toxin Cry3A form oligomers in solution. The structural results presented here indicate that the interactions between domains I and III could be responsible for the initial higher order structure and have implications for the biological activity of these toxins. There are seven additional single amino-acid residues in the sequence of Cry3Bb1 compared with that of Cry3A; one in domain I, two in domain II and four in domain III, which also shows the largest conformational difference between the two proteins. These changes can be implicated in the selectivity differences noted for these two delta-endotoxins.

Although substitution of tyrosine, phenylalanine, tryptophan, or arginine for leucine 22 in human dihydrofolate reductase greatly slows hydride transfer, there is little loss in overall activity (kcat) at pH 7.65 (except for the arginine 22 variant), but Km for dihydrofolate and NADPH are increased significantly. The greatest effect, decreased binding of methotrexate to the enzyme-NADPH complex by 740- to 28,000-fold due to a large increase in the rate of methotrexate dissociation, makes these variants suitable to act as selectable markers. Affinities for four other inhibitors are also greatly decreased. Binding of methotrexate to apoenzyme is decreased much less (decreases as much as 120-fold), binding of tetrahydrofolate is decreased as much as 23-fold, and binding of dihydrofolate is decreased little or increased. Crystal structures of ternary complexes of three of the variants show that the mutations cause little perturbation of the protein backbone, of side chains of other active site residues, or of bound inhibitor. The largest structural deviations occur in the ternary complex of the arginine variant at residues 21-27 and in the orientation of the methotrexate. Tyrosine 22 and arginine 22 relieve short contacts to methotrexate and NADPH by occupying low probability conformations, but this is unnecessary for phenylalanine 22 in the piritrexim complex.

Structural data from two independent crystal forms (P212121 and P21) of the folate (FA) binary complex and from the ternary complex with the oxidized coenzyme, NADP+, and recombinant Pneumocystis carinii dihydrofolate reductase (pcDHFR) refined to an average of 2.15 A resolution, show the first evidence of ligand-induced conformational changes in the structure of pcDHFR. These data are also compared with the crystal structure of the ternary complex of methotrexate (MTX) with NADPH and pcDHFR in the monoclinic lattice with data to 2.5 A resolution. Comparison of the data for the FA binary complex of pcDHFR with those for the ternary structures reveals significant differences, with a >7 A movement of the loop region near residue 23 that results in a new "flap-open" position for the binary complex, and a "closed" position in the ternary complexes, similar to that reported for Escherichia coli (ec) DHFR complexes. In the orthorhombic lattice for the binary FA pcDHFR complex, there is also an unwinding of a short helical region near residue 47 that places hydrophobic residues Phe-46 and Phe-49 toward the outer surface, a conformation that is stabilized by intermolecular packing contacts. The pyrophosphate moiety of NADP+ in the ternary folate pcDHFR complexes shows significant differences in conformation compared with that observed in the MTX-NADPH-pcDHFR ternary complex. Additionally, comparison of the conformations among these four pcDHFR structures reveals evidence for subdomain movement that correlates with cofactor binding states. The larger binding site access in the new "flap-open" loop 23 conformation of the binary FA complex is consistent with the rapid release of cofactor from the product complex during catalysis as well as the more rapid release of substrate product from the binary complex as a result of the weaker contacts of the closed loop 23 conformation, compared to ecDHFR.

A method is presented for producing a seed-stock mixture for macromolecular crystallization. A PTFE bead and micro-centrifuge tube act as mortar and pestle for pulverizing seed crystals of macromolecules. Energy for the bead's motion is supplied by a vortex mixer or an ultrasonic bath. The crushed crystal is serially diluted to prepare a seed-stock mixture of the desired concentration for crystallization. Crystals produced using both hanging-drop vapor diffusion and a capillary microbatch method show expected dilution behavior. This technique of producing seed stock is compared with traditional means and advantages over the standard protocol are demonstrated.

SummaryNucleotide biosynthesis pathways have been reported to be essential in some protozoan pathogens. Hence, we evaluated the essentiality of one enzyme in the pyrimidine biosynthetic pathway, dihydroorotate dehydrogenase (DHODH) from the eukaryotic parasite Trypanosoma brucei through gene knockdown studies. RNAi knockdown of DHODH expression in bloodstream form T. brucei did not inhibit growth in normal medium, but profoundly retarded growth in pyrimidine-depleted media or in the presence of the known pyrimidine uptake antagonist 5-fluorouracil (5-FU). These results have significant implications for the development of therapeutics to combat T. brucei infection. Specifically, a combination therapy including a T. brucei-specific DHODH inhibitor plus 5-FU may prove to be an effective therapeutic strategy. We also show that this trypanosomal enzyme is inhibited by known inhibitors of bacterial Class 1A DHODH, in distinction to the sensitivity of DHODH from human and other higher eukaryotes. This selectivity is supported by the crystal structure of the T. brucei enzyme, which is reported here at a resolution of 1.95 Å. Additional research, guided by the crystal structure described herein, is needed to identify potent inhibitors of T. brucei DHODH.