Justin M. Kollman

A previous report identified and classified a small family of gram-negative bacterial drug and heavy metal efflux permeases, now commonly referred to as the RND family (TC no. 2.6). We here show that this family is actually a ubiquitous superfamily with representation in all major kingdoms. We report phylogenetic analyses that define seven families within the RND superfamily as follows: (1) the heavy metal efflux (HME) family (gram negative bacteria), (2) the hydrophobe/amphiphile efflux-1 (HAE1) family (gram negative bacteria), (3) the nodulation factor exporter (NFE) family (gram negative bacteria), (4) the SecDF protein-secretion accessory protein (SecDF) family (gram negative and gram positive bacteria as well as archaea), (5) the hydrophobe/amphiphile efflux-2 (HAE2) family (gram positive bacteria), (6) the eukaryotic sterol homeostasis (ESH) family, and (7) the hydrophobe/amphiphile efflux-3 (HAE3) family (archaea and spirochetes). Functionally uncharacterized proteins were identified that are members of the RND superfamily but fall outside of these seven families. Some of the eukaryotic homologues function as enzymes and receptors instead of (or in addition to) transporters. The sizes and topological patterns exhibited by members of all seven families are shown to be strikingly similar, and statistical analyses establish common descent. Multiple alignments of proteins within each family allow derivation of family-specific signature sequences. Structural, functional, mechanistic and evolutionary implication of the reported results are discussed.

A crystal structure of human fibrinogen has been determined at approximately 3.3 A resolution. The protein was purified from human blood plasma, first by a cold ethanol precipitation procedure and then by stepwise chromatography on DEAE-cellulose. A product was obtained that was homogeneous on SDS-polyacrylamide gels. Nonetheless, when individual crystals used for X-ray diffraction were examined by SDS gel electrophoresis after data collection, two species of alpha chain were present, indicating that some proteolysis had occurred during the course of operations. Amino-terminal sequencing on post-X-ray crystals showed mostly intact native alpha- and gamma-chain sequences (the native beta chain is blocked). The overall structure differs from that of a native fibrinogen from chicken blood and those reported for a partially proteolyzed bovine fibrinogen in the nature of twist in the coiled-coil regions, likely due to weak forces imparted by unique crystal packing. As such, the structure adds to the inventory of possible conformations that may occur in solution. Other features include a novel interface with an antiparallel arrangement of beta chains and a unique tangential association of coiled coils from neighboring molecules. The carbohydrate groups attached to beta chains are unusually prominent, the full sweep of 11 sugar residues being positioned. As was the case for native chicken fibrinogen, no resolvable electron density could be associated with alphaC domains.