We have established detailed structural reaction profiles for two enzymes, E. coli cytidine deaminase (CDA) and B. stearothermophilus Tryptophanyl-tRNA synthetase (TrpRS). For each of these, X-ray crystal structures representing different stages of catalysis were solved. Superposition of the active sites reveal molecular rearrangements which can be related to catalysis. For TrpRS, the surprise resides in the observation that the conformational rearrangements are induced almost entirely by the binding of different adenine nucleotide ligands. Particularly interesting are two, quite different ATP complexes, one open and the other closed. In the latter complex TrpRS makes a nearly complete catalog of specific interactions, and the nucleotide triphosphate is entirely enclosed, while in the open complex few of these interactions are observed. Paradoxically, ATP binds more tightly to the open than to the closed state. This implies that the conformational change closing the active site is unfavorable, storing much of the potential binding free energy in destabilizing protein conformation. The three-state behavior of TrpRS is therefore analogous to those of transducing enzymes like myosin and the F1 ATPase.
Tryptophanyl-tRNA synthetase
Retailleau, P., Huang, X. Yin, Y., Hu, M., Weinreb, V., Vachette, P., Vonrhein, C., Bricogne, G., Roversi, P., Ilyin, V., and Carter, C. W. Jr. (2003) “Interconversion of ATP binding and conformational free energies by Tryptophanyl-tRNA Synthetase: structures of ATP bound to open and closed, pre-transition-state conformations”, J. Mol. Biol., 325:39-63. pdf
Retailleau, P., Yin, Y., Hu, M., Roach, J. M., Bricogne, G., Vonrhein, C., Roversi, P., Blanc, E., Sweet, R. M., and Carter, C. W., Jr , (2001) “High Resolution Experimental Phases for Tryptophanyl-tRNA (TrpRS) Synthetase Complexed with Tryptophanyl-5’AMP”, Acta Crystallographica, D57, 1595-1608.pdf
Carter, C. W., Jr., Ilyin, V., Yin, Y., Huang, X., and Retailleau, P. (2001) “Three TrpRS Conformations Stabiltize a Dynamic, Dissociative Transition State” in Using Crystallography to Understand Enzyme Mechanisms, Transactions of the American Crystallographic Association, Inc. 35:19-36.pdf
Praetorius-Ibba, M., Stange-Thomann, N., Kitabatake, M., Ali, K., Söll, I., Carter, C. W., Jr., Ibba, M., and Söll, D., “Ancient Adaptation of the Active Site of Tryptophanyl-tRNA Synthetase for Tryptophan Binding”, Biochemistry, 39:13136-13143. 2000.pdf
Ilyin , V., Temple, B., Hu, M., Li, G.-P., Yin, Y., Vachette, P., & Carter, C. W., Jr., “2.9Å Crystal Structure of Ligand-free Tryptophanyl-tRNA Synthetase: Domain Movements Fragment the Adenine Nucleotide Binding Site” Protein Science 9:218-231. 2000.pdf
Cytidine deaminase
Noonan, Ryan C., Carter, Charles W., Jr., and Badgassarian, Carey K. (2002) “Enzymatic conformational fluctuations along the reaction coordinate of cytidine deaminase”, Protein Science, 11:1424-1434.pdf
Deerfield, D. W. II, Carter, C. W., Jr., and Pedersen, L. G. (2001) Models for Protein-Zinc Ion Binding Sites. II. The Catalytic Sites. Int. J. Quant. Chem. 83:150-165.pdf
Lewis, J. P., Carter, Charles W., Jr., Hermans, Jan, Pan, Wei, Lee, Tai-Sung, and Yang, Weitao, “Active Species for the Ground-State Complex of Cytidine Deaminase: A Linear-Scaling Quantum Mechanical Investigation” J. Am Chem. Soc. 120:5407-5410, 1998.pdf
Our work has produced two different examples of how homology modeling suggests interesting structural relationships that illuminate molecular evolution.
Structural homology of Class II aminoacyl-tRNA synthetases to the HSP70 family and the existence of a gene whose sense and antisense strands code for a dehydrogenase and an HSP70 chaperonin justify reconsideration of a possible sense/antisense ancestry for the two synthetase classes.
Carter, C. W., Jr., and Duax, William L. (2002) “Did tRNA Synthetase Classes Arise on Opposite Strands of the Same Gene?” Molecular Cell, 10:705-708.pdf
The other example involves the relationship between the mammalian mRNA editing enzyme, apolipoprotein B editing cytidine deaminase, APOBEC1, and the E. coli cytidine deaminase. Gaps in the sequence of APOBEC1 amount to almost exactly the mass of the minimal RNA substrate. The gaps eliminate precisely the regions where the E. coli enzyme interacts with the ribose hydroxyl groups, and the remaining homologies suggest a model in which the missing peptide segments leave channels which could represent the path of the RNA substrate. This modeling led to formulation of a sequence profile for Apobec-like proteins. The Scott group (London) has used it to identify many new family members.
Navaratnam, N., Fujino, T., Bayliss, J., Jarmuz, A., How, A., Richardson, N., Angelika, S., Battacharya, S., Carter, C. W., Jr. & Scott, J. E. coli Cytidine Deaminase Provides Molecular Model for ApoB RNA Editing: Implications for Asymmetric RNA Substrate Presentation and Evolution of C to U Editing. J. Mol. Biol. 275:695-714 1998.pdf
Scott, J., Navaratnam, N., and Carter, C. W., “Molecular modeling and the appolipoprotein B containing lipoproteins” Atherosclerosis, 141(suppl 1):S17-S24. 1998.pdf
Scott, J., Navaratnam, N., and Carter, C. W., “Molecular modeling of the biosynthesis of the RNA-editing enyzme APOBEC-1, responsible for generating the alternative forms of appolipoprotein B” Experimental Physiology, 84:791-900. 1999.
Carlow, D. C., Carter, C. W., Jr., Mejlhede, N., Neuhard, J., and Wolfenden, R., “Cytidine Deaminases from B. subtilis and E. coli: Compensating Effects of Changing Zinc Coordination and Quaternary Structure” Biochemistry, 38:12258-12265. 1999.pdf
Statistical Experimental Design
Screening and response surface designs based on randomized sampling have been used to characterize useful multidimensional surfaces in crystal growth and molecular biology.
Yin, Yuhui. and Carter, Charles W., Jr.: "Incomplete Factorial and Response Surface Methods in Experimental Design: Yield Optimization of tRNATrp from in vitro T7 RNA Polymerase Transcription" Nucl. Acids. Res. 24, 1279-1287 1996.pdf
Yin, Y. and Carter, C. W., Jr.: Quantitative Analysis in the Characterization and optimization of Protein Crystal Growth. Acta Crystallographica, Section D50, 572-590 1994.pdf
Bioinformatics and Protein Science
Multivariate modeling has proven useful in the analysis of combinatorial libraries of mutant proteins, and the application of Delaunay tessellation and likelihood scoring derived from the database of known protein structures has provided a good predictive model for analyzing cavity-forming hydrophobic core mutations.
Carter, C. W., Jr., LeFebvre, B., Cammer, S. A., Tropsha, A. & Edgell, M. H. (2001). Four-body potentials reveal protein-specific correlations to stability changes caused by hydrophobic core mutations. Journal of Molecular Biology 311(4):625-638.pdf
Carter, Charles W., Jr., Tropsha, Alex, and Edgell, Marshall. (2002) “Energetics of Enzyme Stability”, Trends in Biotechnology, 20:2-3.pdf
Lahr, S. J., Broadwater, A. Carter, C. W., Jr., Collier, M. Hensley, L., Waldner, J., Pielak, G. J., and Edgell, M. H., “Patterned Library Analysis: A method of the quantitative assessment of hypotheses concerning the determinants of protein structure” Proc Nat. Acad. Sci., USA, 96:14860-14865. 1999.pdf
Crystallographic Phase Determination
The Sayre squaring equations form the basis for an objective function that should be useful in refining phases, but its utility is limited because the global residual in implies distributes discrepancies throughout the unit cell. Reformulation in terms of local squaring functions enables powerful density modification procedures applicable throughout the unit cell. Generalization of these functions to non-spherical templates effectively removes the limitations imposed on the use of the Sayre squaring relation by the requirement of atomic resolution data.
Roach, J. M. and Carter, C. W., Jr. (2003) “Local squaring functions for non-spherical templates” Acta Crystallographica In press:215-220.pdf
Roach, J. M. and Carter, C. W., Jr. (2002) “Local Squaring Equations” Acta Crystallographica A58:215-220.pdf
Roach, J.M., Retailleau, P. and Carter, C. W., Jr. “Phase Determination via Sayre-type Equations with Anomalous Scattering”, Acta Crystallographica, A57:341-350, 2001.pdf