Tsinghua Science and Technology Tsinghua University, China ISSN: 1007-0212 Vol. 6, Num. 3, 2001, pp. 248-252

Tsinghua Science and Technology, Vol. 6, No. 3, August 2001 pp. 248-252

Computational Biology Study of S100 Family with Suggestions for Crystallization*

WANG Yu, YE Sheng, RUAN Ge, M. Bartlam, JIANG Fan, RAO Zihe 

Laboratory of Structural Biology, School of Life Sciences and Engineering, Tsinghua University, Beijing 100084, China

*Supported by the National Natural Science Foundation of China (No.39870174 and 39970155), Project “863” (103130306), and Project “973” (G19999075602, G1999011902, and 1998051105)

Received: 2000-09-15

Code Number: ts01075

Abstract:   

The S100 family is a class of calcium-regulated proteins with EF-hand. They are widely distributed and are implicated in diverse intracellular and extracellular physiological processes. A study of the S100 family using computational biology methods such as multiple sequence alignment, structural alignment and the construction of an evolutionary tree will promote  understanding of S100 protein structures and their function, and could provide suggestions for crystallization.

Key  words:  S100 family; computational biology; sequence alignment; structural alignment; crystallization

Introduction   

Ca²⁺  as a second messenger controls various aspects of cell behavior, in part through interaction with a large member of Ca²⁺  binding proteins with EF-hand. The S100 family is one of the largest subfamilies of Ca²⁺  binding proteins consisting of 17 members which exhibit 25% to 65% homology at the protein level. The S100 proteins are dimers belonging to a structurally related family containing two EF-hand motifs in each polypeptide chain[1]  . The S100 proteins are widely distributed and play important roles in many diverse intracellular and extracellular physiological processes, such as cell generation, extracellular signal conduction, cell motor, tumor invasion and metastasis[2]. However, the general function of S100 proteins is presently  unknown and only 8 protein structures have been reported. Therefore, further research on S100 protein structures is needed to establish the relationship between structure and function.

1 Materials and Methods  

Eight  members of the S100 family with known structures were selected from a search of the PDB database. Their descriptions are listed in Table 1. The monomers with Ca²⁺  binding were used for structural alignment. The Clustal W software package was used to perform multiple sequence alignment[3]. The Insight II software package and the CCP4 program LSQKAB were used for the structural alignment of the eight known S100 structures, and the PHYLIP software package was used to construct an evolutionary tree for the S100 family.

2 Results and Discussion  

2.1 Sequence alignment

The loop region of the N-terminal EF-hand forms the low-affinity calcium-binding site containing 14 amino acid residues, while the loop region of the C-terminal EF-hand forms the high-affinity calcium-binding site containing 12 amino acid residues, which are highlighted in Fig.1. Comparison of the high-affinity calcium-binding site with the low-affinity calcium-binding site shows that the sequence of the high-affinity calcium-binding site which is related to the function of calcium-binding of this S100 family, is more highly conserved, Fig.2. Both human S110 and S107 lack the ability to bind calcium in the N-terminal EF-hands, which is consistent with a shortening of the loop region by three residues[4]. Figure 3 indicates the evolutionary trace of the sequences of these 8 S100 proteins. The numbers on the nodes are the fractions of reliability test using a bootstrapping tool.

2.2 Structural alignment

The overall structures of the S100 proteins are very similar, as shown in Fig.4. Residues in the boxes belong to the structurally conserved regions (SCRs) determined by structural alignment; the highlighted residues form the calcium-binding site. Each monomer contains four helices (Fig.5) or five helices (Fig.6), numbered I, II (and II' for structures with five helices), III and IV. I and II are part of the N-terminal EF-hand, II' is the linker helix, while III and IV are parts of  the C-terminal EF-hand.

As  the S100 family is a class of calcium-regulated proteins with EF-hand, further research on the conformation of the calcium-binding site is very important for function assignment. The RMS deviation for the N-terminal low-affinity calcium-binding sites was 1.236 (Fig.7); while the RMS deviation for the C-terminal high-affinity calcium-binding sites was 0.815 (Fig.8), which indicates that not only the sequence but also the structure of the high-affinity calcium-binding site is more highly conserved than the low-affinity calcium-binding site. The RMS deviations of the C-terminal high-affinity calcium-binding sites calculated site by site (Fig.9) show that theresidues situated at the edge of the calcium-binding site are more highly conserved than those located in the central part of the site in the structure. This could prove useful for the construction of single point mutants of this S100 family.  

2.3 Crystallization

At present, the structures of four S100 family proteins have been solved by X-ray crystallography, with their crystallization conditions summarized in Table 2. In each case, the preciptant was PEG 4000 and the solution was mildly alkaline in pH. The preciptant concentrations were very similar, for reference for studies of other S100 family protein crystallizations. For example, while the X-ray structures of human S100A1, S100A2, and S100A4 have not yet been solved, their sequences are very similar to the 4 S100 proteins with known X-ray structures and they have the same potential calcium-binding sites (in the boxes, Fig.1). As a result, the previously used crystallization conditions could be used as a guide for the crystallization of other S100 proteins. Crystals of human S100A1 were obtained using the previously reported crystallization conditions as a guide. Data was collected in-house using a Rigaku 18 kW X-ray generator and a MarResearch 345 mm image plate. The data was processed using DENZO and was found to belong to spacegroup P63 with unit cell dimensions a=b=5.2290 nm, c=10.4674 nm, a=b=90°, and g=120°.

References

1. Fiona E M Gibbs, Mark C Wilkinson, Philip S Rudland, et al. Interactions in vitro of p9Ka, the rat S-100-related, metastasis-inducing, calcium-binding protein. J Biol Chem, 1994, 269(29):  18 992-18 999.
2. Bryony H Lloyd, Angela Platt-Higgins, Philip S Rudland, et al. Human S100A4 (p9ka) induces the metastatic phenotype upon benign tumour cells. Oncogene, 1998, 17: 465-473.
3. Thompsan J D, Higgins D G, Gibson T J. Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 1994, 22:  4673-4680.
4. Brodersen D E, Nyborg J, Kjeldgaard M. Zinc-binding site of an S100 protein revealed.  Two crystal structures of Ca²⁺ -bound human psoriasin (S100A7) in the Zn²⁺ -loaded and Zn²⁺ -free states. Biochemistry, 1999, 38: 1695-1704. 

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