University of Virginia Health System

Email Address: rhk5i@virginia.edu

With Norio Matsushima, Sapporo, we have evaluated the occurrences and characteristics of over 1000 tandem repeats, as found in over 10% of all known proteins. In collaboration with Sven Hovmöller (Physical Chemistry, Stockholm,Sweden) and Roger Ison (Mantic Software - Loveland, CO) we have initiated a knowledge based program to predict ab initio the tertiary structures of proteins from their amino acid sequences. Many tandem repeats do not have a single conformation under physiological conditions; this is consistent with the distribution of structures generated by prediction.

We have completed an extensive analysis of the evolution of 1000 EF-hand homolog proteins and identified 78 distinct subfamilies. Some subfamilies, such as calmodulin, troponin C, essential light chains, and regulatory light chains of myosin, are congruent; all evolved from a single protein that contained four EF-hand domains. Others, like calpain, evolved from a single EF-hand domain by recent gene duplications and fusions. Most other subfamilies have complex evolutionary histories involving multiple gene translocations and splicings. Intron sites are found at many positions within domains and appear not to have facilitated the multiple domain swappings involved in the evolution of this complex subfamily.

We have determined the crystal structures of DAHPS and of KDOPS in various liganded states. Comparisons and evaluations of these structures have revealed the coordination of PEP in the homologous enzymes, the twisting from planarity of the PEP by the enzymes as an apparent initial step in the reaction of PEP with E4P (in DAHPS) or with A5P (in KDOPS). The coordinations of E4P and of A5P have also been determined as have their initial interactions with PEP in the first step of condensation. The role of Mn 2+ in coordinating PEP in DAHPS is explained, as is the lack of metal binding in KDOPS with compensatory coordination of PEP by Lys and Arg. We have determined the crystal structure of DAHPS complexed with its cognate feedback inhibitor, Phe, and defined the specificity and mechanism of feedback inhibition. The absence of feedback inhibition in KDOPS is rationalized. We have determined the structure of DAHPS from Thermatoga maritima. It contains an extra domain, homologous to the metal chaperone, at the N-terminus; this provides a Me 2+ binding site and is not involved in dimerization. T. maritima DAHPS is subject to inhibition by Phe; however, the loop comprising the Phe binding site in the E. coli DAHPS is not present in T. maritima. DAHPS (Tm) has evolved a completely different mechanism of feedback inhibition.