Aplysia temptin - the 'glue' in the water-borne attractin pheromone complex.
Temptin, a component of the complex of water-borne protein pheromones that stimulate attraction and mating behavior in the marine mollusk Aplysia, has sequence homology to the epidermal growth factor (EGF)-like domains of higher organisms that mediate protein-cell surface contact during fertilization and blood coagulation. In this work, recombinant temptin for structural and functional studies was produced in Escherichia coli using a cold shock promoter and purified by RP-HPLC. CD spectra confirmed a predominantly beta-sheet structure. Two disulfide bonds were determined via limited proteolysis and MS. One internal disulfide (Cys57-Cys77) was predicted from initial alignments with class I EGF-like domains; the second, between Cys18 and Cys103, could protect temptin against proteolysis in seawater and stabilize its interacting surface. A three-dimensional model of temptin was prepared with our MPACK suite, based on the Ca(2+)-binding, EGF-like domain of the extracellular matrix protein fibrillin. Two temptin residues, Trp52 and Trp79, which align with cysteine residues conserved in fibrillins, lie adjacent to and could stabilize the disulfide bonds and a proposed metal-binding loop. The water-borne pheromone attractin in egg cordon eluates is complexed with other proteins. Docking results with our model and the NMR structure of attractin suggest that one face of temptin interacts with the pheromone, perhaps controlling its access to the cellular receptors. Gel shifts confirmed that temptin complexes with wild-type attractin. These results indicate that temptin, analogous to the role of fibrillin in controlling transforming growth factor-beta concentration, modulates pheromone signaling by direct binding to attractin.
Cummins
Scott F
SF
Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX 77555-0857, USA.
Xie
Fang
F
de Vries
Melissa R
MR
Annangudi
Suresh P
SP
Misra
Milind
M
Degnan
Bernard M
BM
Sweedler
Jonathan V
JV
Nagle
Gregg T
GT
Schein
Catherine H
CH
D000595:Amino Acid Sequence; D000818:Animals; D001048:Aplysia / Q000187:drug effects / Q000502:physiology*; D015152:Blotting, Northern; D002942:Circular Dichroism; D004220:Disulfides / Q000378:metabolism; D006023:Glycoproteins / Q000502:physiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010675:Pheromones / Q000494:pharmacology*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D012724:Sex Attractants / Q000494:pharmacology*
D004220:Disulfides; D006023:Glycoproteins; D010675:Pheromones; D012724:Sex Attractants