The solution structure of human calcium-bound S100A4 mutated at four cysteine loci (original) (raw)

2015, Journal of biomolecular NMR

The S100 proteins constitute the largest family within the EF-hand protein superfamily, shown to bind and control various proteins involved in several cellular functions such as proliferation, differentiation, apoptosis, Ca 2? homeostasis, and energy metabolism (Donato et al. 2013; Hermann et al. 2012; Leclerc and Heizmann 2011). They regulate a wide range of important cellular processes via protein-protein interactions (Schafer and Heizmann 1996). Calcium interactions with the EF-hand motifs result in a conformation change of target protein binding by exposing hydrophobic regions in S100 proteins (Smith and Shaw 1998). The calcium binding EF-hand motif starts the action with structural changes in the S100 proteins, allowing them to interact through target selectivity (Yap et al. 1999; Zimmer and Weber 2010). The protein S100A4 was first derived from both tumor and stroma. It is a homodimeric protein in solution and has been shown to function as a metastasis-promoting protein (Ambartsumian et al. 2005; Ismail et al. 2010). Its presence has now been well documented in many cancers including breast, colorectal, gastric, pancreatic, and bladder cancers. It plays a role in tumor formation and angiogenesis (Ambartsumian et al. 2001; Barraclough et al. 2009; Ford and Zain 1995). The hinge region and C-terminal EF-hand of the S100A4 protein are unique compared to other S100 proteins; however most S100 proteins are involved in target protein binding. The binding of calcium ions produces conformational changes in proteins resulting in exposure of the hydrophobic pocket of residues in helices 3 and 5, the hinge region, and the C-terminal EF-hand (Malashkevich et al. 2008; Mishra et al. 2012; Semov et al. 2005). Previous results have provided insights into the dynamic mechanism of the C-terminal in S100A4 as a mediator of S100A4-driven metastasis, and they highlight its role in tuning the Ca 2?-binding affinity of S100A4. These results also suggest that locking the C-terminus to the core domain may be an alternative strategy for inhibiting its metastasispromoting activities (Duelli et al. 2014). In this study, we mutated four cysteine residues (Cys3, Cys76, Cys81, and Cys86) on S100A4 to serine. The Cys3 residue is located very near the N-terminus, and the remaining three are in helix 4 (H4) (Pathuri et al. 2008). We labeled this protein 'mutant S100A4' and determined the three-dimensional structure of the calcium-bound of this protein using NMR. With this structure solved, we could explain why full length S100A4 has weaker calcium-binding affinity than its truncated form, where the last 13 amino acids in the C-terminal are deleted. Methods and results Protein expression and purification Wild type human S100A4 contains four cysteine residues. One is at the N-terminal position of the amino acid sequence, and the remaining three are in the H4 region in the presence of DTT as a reducing agent, necessary in NMR buffer conditions. To study the three-dimensional structure Electronic supplementary material The online version of this article (