SPARC (PDB ID: 2V53)
Created by Pratik Patel
SPARC (secreted protein acidic and rich in cysteine) is a collagen-binding glycoprotein that is located in the extracellular matrix (1). SPARC (PDB ID code 2V53), unlike its ligand collagen, does not serve a structural role but rather, it mediates cell-matrix interactions through tissue renewal and tissue remodeling (2). Others have also found it to be involved in embryonic development in invertebrates and a cell-cycle inhibitor (3). In bone, 2V53 is omnipresent and studies with SPARC knock-out mice have displayed bone loss; thus, implying a function for 2V53 in bone development and maintenance (2).
One of SPARC's most important biological functions is tissue homeostasis since it is expressed at high levels in remodeling tissues and has been thought to modulate growth factor activity. In adults, SPARC is highly expressed in remodeling tissues which are healing from a wound or disease. Thus, investigation of SPARC's effects in these remodeling tissues could shed light on potential remedies for illnesses such as cancer and diabetes (2).
SPARC is also referred to as osteonectin, BM-40 and 43K protein. Other members of the same class of proteins as SPARC are thrombospondins (TSP) 1 and 2, osteopontin (OPN), SC1, and QR1. QR1 has been known to be involved in cell-cycle inhibition and another similar protein, hevin, has a similar function as a counteradhesive protein. SC1 is a related protein that has 634 amino acids and shares 53% identity with SPARC, which is 303 amino acids (2). It also shares around 25% of the acidic region of SPARC as well as 60% of the extracellular calcium binding domain. SPARC is acidic and rich in the amino acid cysteine which means there are many disulfide bridges. The isoelectric point is calculated to be around 4.73 according to the ExPASy Proteomics Server. It's secondary structure consists of 7 alpha helices, 6 beta sheets, and many random coils. A similar protein to SPARC, calcyphosin (PDB ID: 3E3R), contains 11 helices and 4 beta strands. 3E3R is about 57% similar to SPARC and also serves as a calcium binding protein (2). Another similar structure is calmodulin (PDB ID: 1EXR) which is another calcium binding protein. SPARC and calmodulin are significantly significant (p-value of 1.41e-2) and thus some of their functions overlap as well. The secondary structures are not as similar as calmodulin has only 7 helices and 4 beta strands.
SPARC (PDB ID: 2v53) is a 303 amino acid long protein. The first 17 amino acids are the signal sequence which becomes cleaved to produce a 286 amino acid secreted SPARC protein (1). SPARC is divided into four domains. The first of these domains is Domain I which is comprised of amino acids 3-51 of the secreted protein. This region is highly acidic and binds 5-8 calcium ions with a relatively low affinity (Kd: 10e-3-10e-5) (1).
Domain II is comprised of amino acids 52-132 and includes the cysteine rich region with 10 cysteine residues. Amino acids 54-76 comprise the follistatin-like region which contains the homologous to the domain in follistatin (1). There are also two Cu2+ binding sites within this domain and a ( K)GHK sequence at amino acids 120-123 that encodes an angiogenic peptide when released (4). Both of these are implicated in the regulation of cell proliferation. In this domain, N-71 and N-99 are the sites for N-linked glycosylation (5). This domain is an elongated structure consisting of an N-terminal beta-hairpin and a small core of mixed alpha/beta structure (2). The mixed core is composed of a pair of antiparallel alpha-helices connected to three-stranded antiparallel beta-sheet. The hairpin interacts with the core around the disulfide bonds 60-76 and 78-113 and hydrophobic contacts between V-65 and Q-79 (6). The amino acids 72-134 comprise the Kazal-like protease inhibitor region. The remaining disulfide bonds in this domain are amino acids 55-66, 84-106, and 95-132 (6).
Domain III is comprised of amino acids 133-227 with most of it as an extended series of alpha-helical segments. It contains an endogenous protease-sensitive site for extracellular interactions.C-138 at the N-terminus of this region is disulfide bonded to C-248 of domain IV (6). The extensive alpha-helical structure is calcium dependent (1). Affinity for collagen IV, one of SPARC's ligands, is increased by cleavage of the peptide bond between L-197 and L-198. The amino acids within this domain that are required for collagen I and IV binding are R-149 and N-156 (2).
Collagen III binding is showcased here with the triple helix of collagen III, each helix consisting of 33 amino acids. The three chains are known as the leading, middle, and trailing chains because each is staggered by one amino acid. The middle and trailing chains are the only ones involved in binding to SPARC. Only the EC domain (amino acids 204-286) of SPARC interacts in collagen III binding. The binding site involves the long alphaA helix and the adjacent alphaE-F helices loop, which together forms a pocket known as the Phe pocket (3). This pocket forms the pocket for F23 of the trailing chain of collagen which is bordered by apolar SPARC residues of F-146, M-150, W-153, and L-242 and a salt bridge between R-149 and E-246. The phenyl ring of F-23 interacts with W-153. The second major interaction with SPARC is with the M-21 of the middle chain and W-153 and L-242 of SPARC. Lastly, another Phe pocket is made with F-23 of the middle chain and M-245 of SPARC. The residues necessary for binding to collagen III are R-149, L-242, M-245, and E-246 (3).
Domain IV is comprised of amino acids 228-285 and contains an extracellular calcium (EC) module with two EF-hand motifs. This module is globular and interacts with Domain II to stabilize binding of calcium, which makes calcium binding high affinity (2). The disulfide bond between C256-C272 stabilizes the EF-hand-like domain (1). This EF-hand domain is implicated in the calcium dependent binding to collagen IV because domain III's alpha-helical structure is calcium dependent (1). The amino acids crucial for binding for collagen I and IV binding from this domain are L-242, M-245, and E-246 (7). The EC domain inhibits cell spreading and proliferation and enables the protein to bind to cells and matrix (4). The EC module binds to PDGF, which is a ligand of SPARC, but this binding is calcium independent (2).