THE CRYSTALLOGRAPHIC STRUCTURE DETERMINATION OF THE LEUCINE, ISOLEUCINE, VALINE-BINDING PROTEIN FROM ESCHERICHIA COLI
SAPER, MARK AARON
Doctor of Philosophy
Periplasmic binding proteins from gram-negative bacteria are essential components in the osmotic shock-sensitive transport systems for carbohydrates, amino acids, and ions. The structure of the leucine, isoleucine, valine-binding protein (LIV-BP), an integral part of the high affinity branched-chain, aliphatic amino acid transport system in Escherichia coli, has been determined by X-ray crystallography. Purified LIV-BP (M(,r) = 36,770) that was devoid of residually-bound amino acid formed large, polyhedral crystals from 12% polyethylene glycol at pH 4.5. The structure was solved by the multiple isomorphous replacement method at 3.0 (ANGSTROM) resolution using five heavy atom derivatives (mean figure of merit = 0.77 for 6653 reflections). A polyalanine model was fit to the electron density map with the modeling program FRODO implemented on an Evans & Sutherland PS 300 interactive graphics system. The model of the polypeptide chain backbone reveals two distinct, globular domains connected by three strands. Between the domains is a wide cleft which is accessible to solvent. Though lacking sequence similarity, each of the two domains has essentially the same secondary structure arrangement: a central, five-strand parallel (beta)-sheet flanked by two major (alpha)-helices on either side running anti-parallel to the sheet. The secondary structure elements are connected in the classic (beta)(alpha)(beta) folding pattern. The amino acid binding site was located by soaking crystals in solutions of 10 or 50 mM L-leucine, L-isoleucine, L-valine, or L-threonine. Difference Fourier maps in all cases displayed a significant peak located in the cleft between the two domains, but confined only to the wall of the N-terminal domain near two loops that connect (beta)-sheets with helices. Despite little sequence homology, the supersecondary structure of the LIV-BP domains is very similar to the corresponding domains of the bilobate L-arabinose-binding protein (ABP). Seventy percent of the (alpha)-carbons from the two proteins were determined to be equivalent. The major difference between the two structures is the orientation of the two domains and the size of the cleft: narrow in ABP and wide-open in LIV-BP. Since the ABP crystal structure contains bound substrate and LIV-BP doesn't, a substrate-induced cleft closure is proposed. The LIV-BP structure with an open cleft may be typical of all unliganded binding proteins.