Role of arginine-163 and the 163REEK166 motif in the oligomerization of truncated alpha A-crystallins.
To gain insight into the mechanism by which Arg-163 influences oligomerization of alphaA-crystallin, we prepared a series of truncated alphaA-crystallins with or without mutation of the Arg-163 residue. Expression of the proteins was achieved in Escherichia coli BL21 (DE3) pLysS cells, and alphaA-crystallin was purified by size-exclusion chromatography. Molecular mass was determined by molecular sieve HPLC, chaperone activity was assayed with alcohol dehydrogenase as the target protein, and structural changes were ascertained by circular dichroism (CD) measurements. With an increasing number of residues deleted, there was about a 3% decrease in oligomeric size per residue, until 10 residues were deleted. When 11 residues, including Arg-163, were deleted, the oligomeric size decreased 85%. Mutation of Arg-163 to Gly (R163G) did not affect the molecular mass in the full-length alphaA-crystallin. However, R163G mutants of all the truncated alphaA-crystallins showed a decrease in oligomeric size, those lacking 8, 9, and 10 residues showing 60-80% decrease and those lacking 5, 6, and 7 residues showing only a 7-14% decrease as compared to the corresponding truncated alphaA-crystallin. These data suggest that R163, E164, E165, and K166 in the REEK motif are also relevant to alphaA-crystallin oligomerization. The molecular masses of alphaA1-163 and alphaA1-163 (R163K) were nearly the same, which suggests that the role of Arg-163 is to provide a positive charge for intersubunit electrostatic interactions in the C-terminal domain. In alphaA1-162 (S162R), recovery of the molecular mass to the level in alphaA1-163 has not occurred; this shows that the actual position of R163 is important.
Rajan
Shanthi
S
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205-7199, USA.
Chandrashekar
Reena
R
Aziz
Atya
A
Abraham
Edathara C
EC
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry*; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017384:Sequence Deletion / Q000235:genetics; D055672:Static Electricity; D038202:alpha-Crystallin A Chain / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D018832:Molecular Chaperones; D021122:Protein Subunits; D038202:alpha-Crystallin A Chain; D001120:Arginine