Binding of Manganese Stabilizing Protein to Photosystem II:  Identification of Essential N-Terminal Threonine Residues and Domains that Prevent Nonspecific Binding

• Published in: Biochemistry (Easton) Vol. 42; no. 20; pp. 6193 - 6200
• Main Authors: Popelkova, Hana, Im, Michael M., Yocum, Charles F.
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 27-05-2003
• Subjects: Amino Acid Sequence; Base Sequence; Binding Sites - genetics; Circular Dichroism; DNA, Plant - genetics; Manganese - metabolism; Oxygen - metabolism; Photosynthetic Reaction Center Complex Proteins - chemistry; Photosynthetic Reaction Center Complex Proteins - genetics; Photosynthetic Reaction Center Complex Proteins - metabolism; Photosystem II Protein Complex; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - metabolism; Protein Binding; Protein Structure, Tertiary; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Deletion; Spinacia oleracea - genetics; Spinacia oleracea - metabolism; Threonine - chemistry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0207115

The N-terminus of spinach photosystem II manganese stabilizing protein (MSP) contains two amino acid sequences, 4KRLTYD10E and 15TYL18E, that are necessary for binding of two copies of this subunit to the enzyme [Popelkova et al. (2002) Biochemistry 41, 10038−10045]. To better understand the basis of MSP−photosystem II interactions, the role of threonine residues in the highly conserved motifs T(Y/F)DE and TY has been characterized. Deletion mutants lacking the first 5, 6, 7, and 15 amino acid residues at the N-terminus of the protein were examined for their ability to reconstitute activity in MSP-depleted photosystem II. The results reported here show that truncations of five and six amino acid residues (mutants ΔR5M and ΔL6M mutants) have no negative effect on recovery of oxygen evolution activity or on binding of MSP to photosystem II. Deletion of seven residues (mutant ΔT7M) decreases reconstitution activity to 40% of the control value and reduces functional binding of the mutant protein to photosystem II from two to one copy. Deletion of 15 amino acid residues (mutant ΔT15M) severely impairs functional binding of MSP, and lowers O2 evolution activity to less than 20% of the control. ΔT7M is the only mutant that exhibited neither nonspecific binding to photosystem II nor changes in tertiary structure. These, and previous results, show that the highly conserved Thr7 and Thr15 residues of MSP are required for functional binding of two copies of the eukaryotic protein to photosystem II. Although the N-terminal domains, 1EGGKR6L, 8YDEIQS14K, and 16YL18E of spinach MSP are unnecessary for specific, functional binding interactions, these sequences are necessary to prevent nonspecific binding of the protein to photosystem II.