Human C-reactive Protein (PDB ID = 1GNH)
Created by Jessica Cerullo
Function:
Human C- reactive protein (PDB ID: 1gnh), CRP, is an acute phase serum protein: a protein whose concentration fluctuates in response to cytokines during inflammation (1). CRP is part of the pentraxin family; members of this protein family contain a cyclic pentameric structure and are dependent on calcium for ligand binding (1). CRP has a molecular weight of 230309.25 Da and an isoelectric point of 5.27 (2).
CRP plays a role in innate immunity by activating the complement system, a group of proteins that fight infection by inducing lysis or aptosis of damaged cells and microbes. Injury to tissues stimulates the production of interleukin-6, a cytokine which induces CRP production in the liver and release into the bloodstream (3). Interleukin-1β and tumor necrosis factor-α, both cytokines, also lead to increased production of CRP. In effect, blood CRP levels may serve as an indicator of infection and inflammation (8).
Human C-reactive protein binds to phosphocholine (PCh) in polysaccharides of pathogens and cell membranes of bacteria and other microbes, as well as to monophosphate esters; binding in each case is calcium dependent (3). C1q, a complement system protein, recognizes the CRP-ligand interaction and activates the classical pathway of the complement system. Activation of complement system proteins C3 and C4 enhances phagocytosis by marking the damaged cells or microbes (1). CRP also binds to IgG (an antibody involved in the secondary immune response) Fc-receptors, directly enhancing phagocytosis by leukocytes (8). Phagocytosis helps to clear away dead cells, allowing for the return of normal structure and function (3).
C3a and C5a of the complement pathway stimulate mast cells, which lead to inflammation. Factor H, a regulator of the complement system (5), binds to CRP and blocks the cleavage of C5, reducing the inflammation response. CRP also inhibits the actions of neutrophils, the white blood cells that act as first responders to inflammation (3). As a result, CRP has an anti-inflammatory effect.
Human Serum Amyloid P Component (SAP; PDB ID: 2W08), a structurally similar protein, is another member of the pentraxin family. A BLAST search was performed, and an E-value of 6E-56 (6) indicates similarity in primary structure to CRP. A Z-score of 35.0 and root mean squared deviation of 1.2, as calculated by the DALI Server(7), indicate similarity in tertiary structure. Serum amyloid P component enhances phagocytosis in a similar manner to CRP; the protein binds to Fc receptors of IgG, making the cell more susceptible to phagocytes such as leukocytes. In this way, serum amyloid P component may also be an important protein in innate immunity (4).
CRP is a member of the pentraxin family containing five identical subunits (A-E). The subunits associate non-covalently into a cyclic pentamer, which crystallizes to form a decameric pair. In the crystal structure, the pentamers are staggered, and each subunit is rotated 20 degrees towards the fivefold axis. The rotation places the calcium binding sites of each subunit on the external face (13). In each subunit, 206 residues form 14 anti-parallel β-strands arranged into 2 β-sheets (12). Residues 168-176 form a long α-helix that folds against one of the β-sheets. Residues 43-48 and 186-188 form 310 helical turns. Random coils constitute the remainder of the secondary structure (9). Each subunit contains three salt bridges: Asp-155:Arg-118, Glu-101:Lys-201, and Lys-123:Glu-197 (9). Arg-47 forms a hydrogen bond with both Ser-149 and Ser-151, providing additional stabilization to each subunit (9). The decamer is stabilized by five surface contacts between the pentamers: Thr-173 in each subunit of pentamer 1 interacts with Pro-179 in each subunit of pentamer 2 (13).
CRP depends on calcium for ligand binding. Four of the five subunits in each pentamer bind two calcium ions. Calcium prevents the denaturation of CRP due to high temperatures or high concentrations of urea. In the absence of calcium, CRP can be cleaved at two sites: Asn-145:Phe-146 and Phe-146:Glu-147 (13). Residues 138-150 form a calcium binding loop that undergoes a major conformational change upon the binding of calcium. In calcium-depleted CRP (PDB ID:1LJ7), the 138-150 loop is mobile. When calcium is bound (PDB:1GNH), the loop folds in; this conformational change protects Asn-145:Glu-147 (the site for proteolytic degradation) (14). Glycine residues at positions 143 and 144 provide the loop with the flexibility needed for binding (9). The folded loop places Glu-147 in a position that allows for coordination of the calcium ions. At the calcium binding site, the first calcium ion is coordinated by Asp-60, Asn-61, Glu-138, Asp-140, and Gln-139, whereas the second calcium ion is coordinated by Glu-138, Asp-140, Gln-150, and Glu-147 (9).
In calcium-depleted CRP, conformational changes occur due to crystal packing. The 138-150 loop of one subunit contacts the calcium binding site in a subunit of the other pentamer, resulting in loss of the Arg-47:Ser-149 and Arg-47:Ser-151 hydrogen bonds, as well as movement of the 43-48, 68-72, and 85-91 loops (13). As a result, one subunit of each pentamer is unable to bind calcium (13).
Calcium-bound CRP has a high specificity for ligands with a phosphocholine (PCh) moiety, allowing CRP to bind to the PCh groups of pathogens such as pneumococcal c-polysaccharide, damaged cell membranes, and a number of monophosphate esters (9). The PCh binding site is located adjacent to the calcium binding site. Two phosphate oxygens of the PCh moiety interact directly with the CRP-bound calcium ions (11). The choline group fits into a hydrophobic pocket formed by Phe-66, Leu-64, and Thr-76; Phe-66 interacts directly with the methyl groups of choline (3). Glu-81 further stabilizes binding by interacting with the choline nitrogen. Each calcium-bound subunit has equal affinity for PCh (14). Human C-reactive protein complexed with unconjugated PCh (PDB ID: 1B09) shows little conformational change from calcium-bound CRP. Minor changes occur in the side chain positions involved in binding (11). Since both the hydrophobic pocket and calcium ions are critical for the binding of PCh, the calcium-depleted subunit in each pentamer cannot bind PCh (9).
Both the PCh and calcium binding sites lie on the "recognition" face of the pentamer 2. The "effector" face lies on the opposite side of each subunit and contains a deep cleft that may play an important role in complement activation (9). The cleft extends from the center of each subunit to the internal edge. One end of the cleft is composed of the carboxy end of the 168-176 alpha-helix and a loop composed of residues 177-182. The other end is composed of the amino and caboxy end terminals. The cleft becomes shallow as the pentamer's pore is reached (3). C1q and Fc-receptors bind to CRP on the shallow end of the cleft when CRP is bound to conjugated PCh; the interaction of Fc-recptors with CRP enhances phagocytosis of damaged cells and microbes. Asp-112 and Tyr-175 are contact residues essential to binding C1q, and Glu-188 induces a conformational change in C1q necessary for activation of the complement system (3).
CRP binds a number of ligands that do not contain PCh, including chromatin, histones, galactose containing polysaccharides, and small nuclear ribonucleoprotein particles (snRNPs). Variety in ligand binding suggests that CRP may contribute to an array of metabolic and immune functions (9).
SAP, the structurally similar protein, is also a member of the pentraxin family. SAP contains five identical subunits that crystallize to form a decameric pair in the absence of calcium. The 205 residues of each subunit form a secondary structure consisting of 14 antiparallel β-strands arranged into 2 β-sheets, with an α-helix on the surface of one β-sheet (12). SAP subunits are not tilted with respect to the pentameric axis; the rotation of subunits in CRP is due to a number of loop displacements caused by an insertion of Ser-45-Thr-46 (9). SAP contains Tyr-66 and Lys-79 in place of Phe-66 and Glu-81 (CRP), causing phosphoethanolamine (PE) to be a more favorable ligand than PCh (9). Tyr-76 stabilizes ligand binding by coordinating the PE nitrogen. Since PE is a component of many biological membranes, SAP is able to bind damaged membranes in a similar manner to CRP. With PE bound, SAP mimics CRP by interacting with Fc-receptors to mediate phagocytosis (4) and C1q to activate the classical pathway of complement (15).