W0104

Alanine Dehydrogenase from A. Fulgidus: Forked Path to an Unusual Structure. T. Gallagher, N. Smith, M. Holden, I. Schroeder, and H. Monbouquette, Biotechnology, NIST, 100 Bureau Dr., Gaithersburg, MD 20899 USA.

Macromolecular structure determination offers a series of challenges: crystal growth, diffraction, phasing, map interpretation, and refinement. Although this is their logical sequence, difficult problems often involve backtracking, e.g., when phasing is troublesome, we may rescreen for a new crystal form, effectively starting over. Most proteins that crystallize, do so with several crystal forms;ultimately we want to refine a structure in the highest diffracting form, but this may not be a good form for phasing.

Reversible alanine dehydrogenase (EC 1.1.4.1) activity plays a key metabolic role between the energy-producing Kreb's cycle and the anabolic production of amino acids, and is known to mediate sporulation signaling in many eubacteria. The enzyme in hyperthermophiles is not homologous to its bacterial isofuncts. Instead, it is 30% identical to a human thyroid hormone binding protein, and homologous to widely distributed members of the mu crystallin family.

The structure of the enzyme from Archaeoglobus fulgidus was determined after finding that the crystal form selected for phasing, underwent a change of space group upon binding Ir or Sm (although at different sites!). Despite nonisomorphism, this gave proof of binding, and led to structure determination by MAD. Those crystals were grown by using a pH shift. The structure has been refined in another space group, C2, where resolution was the highest (2.3A), to R=0.20(Rf=0.26), revealing a new fold, details of NAD binding, and providing a model for the human hormone receptor. Although the structure is a dimer, it violates its local dyad symmetry significantly and NCS was not used in refinement. The C2 form was discovered by accident when crystals appeared in unmixed protein droplets beside the main hanging drop. The structure is being refined in all four crystal forms to permit comparison of Ir and Sm binding and analysis of the rearrangements underlying the fortuitous space group change.