| Surfactant protein D (SP-D) plays an important role in defending against pulmonary pathogens by binding to carbohydrate surface features. A preferred binding target of SP-D on Gram-negative bacteria is the core oligosaccharide (OS) region of lipopolysaccharide (LPS). Crystallographic complexes of the neck and carbohydrate recognition domain (NCRD) of human SP-D with six different components of OS have been solved to elucidate the mechanism of interaction. Previously published structures of SP-D complexes with sugars have demonstrated binding interactions between calcium and two adjacent oxygens on the primary sugar ring. However, the present studies show that the heptose common to many different LPS inner core structures, L-glycero-alpha-D- manno-heptose (L,D-Heptose), binds by coordinating calcium through the glyceryl 6-OH and 7-OH groups rather than the sugar ring. Since the ring OH groups of the LPS heptoses are commonly involved in glycosidic or other linkages, the role we have shown for the 6-OH and 7-OH groups provides new insights into how SP-D recognizes Gram-negative bacteria.;Murine models are commonly used experimentally to evaluate in-vivo SPD actions. However, human SP-D has different preferences in carbohydrate binding. For instance, human SP-D prefers N-acetylmannosamine (ManNAc), whereas murine SP-D prefers myoinositol. To help understand these differences we have used a mutant form of human NCRD, D324N/D325N/R343K mutant (NCRD NNK), designed to resemble aspects of the murine protein. Crystal structures of NCRD NNK with myoinositol and ManNAc indicate structural bases for the preferences.;Structures also have been determined of two transcription regulators, Rob and MarR, involved in the development of multiple antibiotic resistance (MAR) in E. coli. Rob contains a C-terminal domain, which is presumed to be involved in the binding of ligands and is unique in this class of regulators. The crystal structure of Rob was determined at 1.9 A. Chenodeoxycholate (CDC) and decanoate (DEC) were shown by intrinsic fluorescence spectroscopy to bind to Rob, and binding affinities were estimated. Models for the binding of CDC or DEC to Rob are proposed based on studies using AUTODOCK.;Crystal structures also were determined of two superrepressor MarR mutants, G95S and D26N, revealing the features likely to underlie the superrepressor characteristics. |
