Two-component systems, the predominant signal transduction strategy used by prokaryotes, involve phosphorelay from a sensor histidine kinase (HK) to an intracellular response regulator protein (RR) that typically acts as a transcription regulator. RRs are modular proteins, usually composed of a conserved regulatory domain, which functions as a phosphorylation-activated switch, and an attached DNA binding effector domain. The crystal structure of a Thermotoga maritima transcription factor, DrrD, has been determined at 1.5 A resolution, providing the first structural information for a full-length member of the OmpR/PhoB subfamily of RRs. A small interdomain interface occurs between alpha 5 of the regulatory domain and an antiparallel sheet of the effector domain. The lack of an extensive interface in the unphosphorylated protein distinguishes DrrD from other structurally characterized multidomain RRs and suggests a different mode of interdomain regulation.

The predicted models for the structures and mode of hormone binding of the glycoprotein hormone receptors are to a large extent consistent with currently available biochemical and mutational data. Repeated sequences in beta-barrel proteins are shown to have general implications for constraints on structure. Averaging techniques used here to recognize the structural motif in these receptors should also apply to other proteins with repeated sequences.

Histidine protein kinases and response regulators form the basis of phosphotransfer signal transduction pathways. Commonly referred to as two-component systems, these modular and adaptable signaling schemes are prevalent in prokaryotes. Structures of the core domains of histidine kinases reveal a protein kinase fold different from that of the Ser/Thr/Tyr protein kinase family, but similar to that of other ATP binding domains. Recent structure determinations of phosphorylated response regulator domains indicate a conserved mechanism for the propagated conformational change that accompanies phosphorylation of an active site Asp residue. The altered molecular surface promotes specific protein-protein interactions that mediate the downstream response.

Formation of local structure and overall chain dimensions in the 124-residue, four-disulfide protein bovine pancreatic ribonuclease A (RNase A) under conditions favoring either the native or partially folded states have been studied by nonradiative excitation energy transfer measurements. Three RNase A derivatives, doubly labeled with 2-naphthylalanine amide (fluorescent donor) at the C-terminus of each and 7-carboxymethylamino-4-methyl-coumarin (fluorescent acceptor) at the epsilon-amino group of lysine 1, 61, and 104, respectively [(1-124)RNase A, (61-124)RNase A, and (104-124)Rnase A], were prepared. RNase A was modified by a two-step labeling strategy involving prior modification of the C-terminus with the donor probe by enzymatic methods, followed by modification of lysine epsilon-amino groups with the coumarin derivative. The derivatives were purified by liquid chromatography and characterized by tryptic mapping. The mono-labeled donor derivative (without acceptor) undergoes a reversible thermal folding transition (Tm = 48.3 degrees C; native RNase A, Tm = 54.4 degrees C), and all labeled derivatives retain enzymatic activity (activities against the substrate cCMP relative to native are 87 +/- 5%, 94 +/- 6.5%, 79 +/- 10%, and 207 +/- 15% for the donor-only and doubly-labeled derivatives with the acceptor at Lys 104, 61, and 1, respectively), supporting the suitability of these derivatives for protein folding studies. Time-resolved fluorescence measurements were used to determine the extent of nonradiative excitation energy transfer between donor and acceptor probes, which allowed recovery of parameters describing the distribution of interprobe distances and the diffusion coefficient of the ends of the segments defined by the pairs of sites labeled by the probes. Use of a donor with a relatively long intrinsic fluorescence decay rate allowed greater precision in the recovery of the interprobe diffusion coefficients compared with earlier studies using donors with shorter intrinsic decay rates, and this parameter provides an important measure of the extent of folding and degree of packing of the chain segments. Analyses for each derivative were carried out under solution conditions favoring native (pH 5.0, 22 degrees C, < 0.7 M guanidinium hydrochloride) or denatured (> 6 M guanidinium hydrochloride) chain conformations, both with and without intact disulfide bonds (in the absence or presence of dithiothreitol, respectively).(ABSTRACT TRUNCATED AT 400 WORDS)

Humanfollicle-stimulating hormone receptor (hFSHR) belongs to family I of G protein-coupled receptors. FSHR extracellular domain (ECD) is predicted to have 8 -9 ␣␤ or leucine-rich repeat motif elements. The objective of this study was to identify elements of the FSHR ECD involved in ligand binding. Preincubation of recombinant hFSHR ECD with rabbit antisera raised against synthetic peptides of hFSHR ECD primary sequence abolished follitropin binding primarily in the region of amino acids 150 -254. Accessibility of hFSHR ECD after hormone binding, captured by monoclonal antibodies against either ECD or FSH, was decreased for the region of amino acids 150 -220 but additionally for amino acids 15-100. Thus, when hFSH bound first, accessibility of antibody binding was decreased to a much larger extent than if antibody was bound first. This suggestion of a conformational change upon binding was examined further. Circular dichroism spectra were recorded for purified single chain hFSH, hFSHR ECD, and hFSHR ECD-single chain hFSH complex. A spectral change indicated a small but consistent conformational change in the ECD⅐FSH complex after hormone binding. Taken together, these data demonstrate that FSH binding requires elements within the leucinerich repeat motifs that form a central region of hFSHR ECD, and a conformational change occurs upon hormone binding. Follicle stimulating hormone (FSH),1 together with luteinizing hormone (LH), thyroid stimulating hormone, and chorionic gonadotropin, belongs to the group of pituitary or placental glycoprotein hormones (GPH). Members of this group belong to the cysteine knot fold family. A common ␣-subunit and a hormone-specific ␤-subunit, each sharing the same fold, form the functional heterodimer. FSH plays a central role in the regulation of mammalian reproduction. In females, it is essential for ovarian and follicular development and maturation, whereas in males it regulates spermatogenesis (1, 2).FSH binds specifically to follicle-stimulating hormone receptors (FSHR) on granulosa cells in the ovary or Sertoli cells in the testis. Binding of FSH to its receptor induces signal transduction by stimulation of adenylate cyclase, which leads to a specific cell response through protein kinase A-dependent pathways (3).The FSHR belongs to the family of G protein-coupled receptors and consists of an uncharacteristically large extracellular domain (ECD) and a seven-pass helical transmembrane domain (reviewed by Simoni et al. (1)). Sequence analysis suggests that the ECD of the glycoprotein hormone receptors (GPHR) contains 8 -9 imperfect leucine-rich repeats (LRR) like the porcine ribonuclease inhibitor (RI), a member of the LRR family. Crystal structure analysis showed that RI consists of 15 LRR, which have a ␤-␣ hairpin unit structure, where the units are aligned parallel to a common axis (4). The resulting structure resembles a horseshoe with ␤-strands forming the inner circumference, whereas the ␣-helices form the outer circumference. This kind of structure shows high flexibility in...

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