Design of high-affinity S100-target hybrid proteins.
S100B and S100A10 are dimeric, EF-hand proteins. S100B undergoes a calcium-dependent conformational change allowing it to interact with a short contiguous sequence from the actin-capping protein CapZ (TRTK12). S100A10 does not bind calcium but is able to recruit the N-terminus of annexin A2 important for membrane fusion events, and to form larger multiprotein complexes such as that with the cation channel proteins TRPV5/6. In this work, we have designed, expressed, purified, and characterized two S100-target peptide hybrid proteins comprised of S100A10 and S100B linked in tandem to annexin A2 (residues 1-15) and CapZ (TRTK12), respectively. Different protease cleavage sites (tobacco etch virus, PreScission) were incorporated into the linkers of the hybrid proteins. In situ proteolytic cleavage monitored by (1)H-(15)N HSQC spectra showed the linker did not perturb the structures of the S100A10-annexin A2 or S100B-TRTK12 complexes. Furthermore, the analysis of the chemical shift assignments ((1)H, (15)N, and (13)C) showed that residues T102-S108 of annexin A2 formed a well-defined alpha-helix in the S100A10 hybrid while the TRTK12 region was unstructured at the N-terminus with a single turn of alpha-helix from D108-K111 in the S100B hybrid protein. The two S100 hybrid proteins provide a simple yet extremely efficient method for obtaining high yields of intact S100 target peptides. Since cleavage of the S100 hybrid protein is not necessary for structural characterization, this approach may be useful as a scaffold for larger S100 complexes.
Rezvanpour
Atoosa
A
Department of Biochemistry, University of Western Ontario, London, Ontario, Canada.
Phillips
Jeremy M
JM
Shaw
Gary S
GS
D000595:Amino Acid Sequence; D000818:Animals; D017306:Annexin A2 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D051347:CapZ Actin Capping Protein / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D020832:EF Hand Motifs; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D050506:Mutant Chimeric Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D009414:Nerve Growth Factors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D011817:Rabbits; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D000099248:S100 Calcium Binding Protein A10
D017306:Annexin A2; D051347:CapZ Actin Capping Protein; D050506:Mutant Chimeric Proteins; D009414:Nerve Growth Factors; D010455:Peptides; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; D000099248:S100 Calcium Binding Protein A10