Molecular cloning and characterization of a tobacco calmodulin binding protein

Abstract

Calmodulin-binding proteins (CaMBP) play important roles in Ca2+ICaMmediated cellular activities. While many CaMBPs have been isolated and characterized in animals, very little is known about plant calmodulin-binding proteins (CaMBPs). A cDNA clone, pTCB 60, encoding a recombinant tobacco CaMBP was isolated previously by screening a tobacco (Nicotiana tabacum L. cv Wisconsin 38) cDNA library using S35_ labeled CaM as a ligand probe. The cDNA clone contained a 1572 bp cDNA insert with an 1184 bp open reading frame encoding 393 amino acid residues. Northern blot analysis of tobacco total RNA with radiolabeled cDNA insert as a probe detected a 2.4 kb mRNA, indicating that pTCB60 is not a full length cDNA clone. The presence of a poly (A) tail at 3' end and the lack of an initiation methionine codon at the beginning of the open reading frame (ORF) of the cDNA sequence, suggest that approximately 830 bases from the 5'end of the message are missing in pTCB60 cDNA clone. The complete sequence of the messenger was determined by two approaches. First, the 800 bp unknown 5'downstream region of the messenger was synthesized and amplified by a slightly modified 5'RACE (rapid amplification of cDNA ends) protocol, cloned and sequenced. Second, a tobacco cDNA library was constructed in Agt11 and rescreened with pTCB60 cDNA probe to isolate a full-length clone. The combined nucleotide sequence of the 5'RACE clone and pTCB60 partial cDNA clone indicated the full length cDNA sequence is 2409 bp containing a 1656 bp ORF encoding 551 amino acid residues. Database searches revealed no similarity with known gene or protein sequences. The 48.5 kD recombinant tobacco CaMBP encoded by pTCB60 cDNA clone was purified by a two step procedure using CaM-Sepharose chromatography and Protosorb immunoaffinity chromatography. Polyclonal antisera raised against the purified recombinant protein recognized a 60 kD polypeptide in western blots of tobacco cell extracts. Northern blot and immunoblot analyses showed differential expression of TCB60 mRNA and the corresponding protein during tobacco cell culture growth and heat shock response. The western blot of tobacco leaf, stem and root proteins indicated the expression of the protein only in leaf tissues. Secondary structure analysis of the deduced amino acid sequence of the recombinant CaMBP suggested the presence of a basic amphiphilic a-helix (BAA) motif at the C-terminus of the protein. A synthetic peptide was made corresponding to the putative BAA motif spanning amino acids 520-538 and its interaction with CaM was analyzed by a variety of methods. Calmodulin exhibited a Ca2+-dependent mobility shift upon binding the synthetic peptide in 4 M urea and native PAGE. The synthetic peptide competitively inhibits CaM-stimulated POE activity (Ki = 15 nM). Upon binding CaM, the fluorescence emission spectra of the peptide containing two tryptophanyl residues shifted toward blue and increased in intensity. The circular dichroism (CD) spectra show the helicity of CaM and peptide increase upon complex formation. IH NMR studies indicate that the peptide interacts with the aromatic residues in the leading helices of domain I and III of CaM. Taken together, these data provide direct evidence that a structurally conserved BAA CaM-binding domain similar to most CaM-binding proteins characterized in animal systems is present in a plant protein.