Sequence and domain organization of scruin, an actin-cross-linking protein in the acrosomal process of Limulus sperm.
The acrosomal process of Limulus sperm is an 80-microns long finger of membrane supported by a crystalline bundle of actin filaments. The filaments in this bundle are crosslinked by a 102-kD protein, scruin present in a 1:1 molar ratio with actin. Recent image reconstruction of scruin decorated actin filaments at 13-A resolution shows that scruin is organized into two equally sized domains bound to separate actin subunits in the same filament. We have cloned and sequenced the gene for scruin from a Limulus testes cDNA library. The deduced amino acid sequence of scruin reflects the domain organization of scruin: it consists of a tandem pair of homologous domains joined by a linker region. The domain organization of scruin is confirmed by limited proteolysis of the purified acrosomal process. Three different proteases cleave the native protein in a 5-kD Protease-sensitive region in the middle of the molecule to generate an NH2-terminal 47-kD and a COOH-terminal 56-kD protease-resistant domains. Although the protein sequence of scruin has no homology to any known actin-binding protein, it has similarities to several proteins, including four open reading frames of unknown function in poxviruses, as well as kelch, a Drosophila protein localized to actin-rich ring canals. All proteins that show homologies to scruin are characterized by the presence of an approximately 50-amino acid residue motif that is repeated between two and seven times. Crystallographic studies reveal this motif represents a four beta-stranded fold that is characteristic of the "superbarrel" structural fold found in the sialidase family of proteins. These results suggest that the two domains of scruin seen in EM reconstructions are superbarrel folds, and they present the possibility that other members of this family may also bind actin.
Way
M
M
Whitehead Institute, Cambridge, Massachusetts 02142.
Sanders
M
M
Garcia
C
C
Sakai
J
J
Matsudaira
P
P
D000177:Acrosome / Q000378:metabolism* / Q000648:ultrastructure*; D000199:Actins / Q000096:biosynthesis* / Q000737:chemistry* / Q000648:ultrastructure; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005260:Female; D015723:Gene Library; D006737:Horseshoe Crabs; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000648:ultrastructure; D017386:Sequence Homology, Amino Acid
D000199:Actins; D018076:DNA, Complementary; D011994:Recombinant Proteins; C083765:scruin protein, Horseshoe Crab