K2 Domain of the Lysine Gingipain (Kgp)
Created by Uma Ayer
The Protein:
Porphyromonas gingivalis is an obligately anaerobic bacterium recognized as an agent of adult periodontitis. Found commonly in the mouth, this bacterium is seen in patients with periodontal disease. P. gingivalis produces cysteine proteinases called gingipains. Gingipains are proteases that break down both proteins and cytokines of Porphyromonas gingivalis. Essentially, gingipains facilitate sustained colonization of P. gingivalis through its inhibitory activation of leukocytes via certain processes (Imamura, 2003). The crystal structure of a domain within the haemagglutinin region of the lysine gingipain, or Kgp, is the structure of interest. Because crystallization for the entire protein has not been done, K2 is the focus of many studies now. The domain was named K2 as it is the second of three homologous structural modules in Kgp. Within the Lysine gingipain, there are three domains, and K2 is the second of three homologous structures in Kgp. The domain consists of 2 subunits, A and B, made entirely out of anti-parallel beta sheets.
Subunit Structures:
A total of 352 residues are present in the K2 domain of the Lysine gingipain. Within the domain, there are two subunits (Subunits A and B) which contain about equal number of residues. Subunit A contains 179 residues while subunit B contains 173 residues. There are also 304 water molecules that surround the protein.The two structures of the K2 domain (designated chains A and B) vary significantly only in their crystal packing arrangements and in interactions with additives such as glycerol (Collyer, 2010).
Secondary structure:
The secondary structure of the cleaved domain of the Lysine gingipain is pretty simple in that it is solely composed of only anti-parallel beta sheets. The Polarity of the protein is also shown, with the yellow representing hydrophobic amino acids (Ala, Val, Phe, Pro, Met, Ili, and Leu); pink representing polar amino acids (Ser, Thr, Tyr, His, Cys, Asn, Gln, Trp, and Gly); blue representing positive charged amino acids (Lys and Arg); and finally, the red representing negatively charged amino acids (Asp and Glu).
Overall, the K2 domain has a ‘jelly-roll’ fold with eleven b-strands forming two anti-parallel b-sheets. These eleven b-strands are linked by ten loops and a one-turn helix which is formed by residues Asn1259-Gly1261 (Collyer, 2010).
Structure and function of Kgp
Within the Lys-gingipain, there are three domains, and K2 is the second of three homologous structures in Kgp. K2 is of special interest, because data indicate that a functional role for K2 is its capacity to enable the "porphyrin auxotroph P. gingivalis to capture essential haem from erythrocytes (Collyer, 2010)." Not too long ago, data confirmed that inactivation of Kgp leads to loss of the black-pigmenting, haemolytic phenotype on blood agar, suggesting that lys-gingipain is involved in the acquisition/storage of haemin, which is probably contributing to accumation of bacteria on the cell surface (Lewis, 1999). According to prior research, the Lysine gingipain is a hemoglobinase which plays a role in heme and iron uptake, affecting the bacterial cell surface (Lewis et al., 1999).
Hemagglutination is a distinctive characteristic of P. gingivalis that discriminates the microorganism from other black-pigmented anaerobic organisms. BecauseP. gingivalis requires heme for growth, hemagglutination serves as the first step in heme acquisition from erythrocytes (Shi et. al, 1999). Mutants defective in Kgp have markedly reduced capacity to sequester haem and are relatively avirulent in animal models and thus, have failed to form black-pigmented colonies caused by hemin accumulation. (Lewis, 1999). Another study has supported the idea that Kgp seems to play an important role in hemin uptake from hemoglobin by the binding, and probably by the subsequent degradation (Kuboniwa, 1998).
Although research has revealed that P. gingivalis produces large quantities of cysteine proteases with either arginine or lysine specificity and that they may may be classified as hemoglobinase due to the degradation of erythrocytes, the role ofLys-gingipain in hemolysis remains unknown. Therefore, the mechanism of Lysine gingipain in the Cysteine protease mechanism is not clear (Lewis, 1999).
Consisting of 27 residues (Thr1270-Trp1296), L8 extends across one end of the b-barrel and then turns around and comes back to the same side to connect to b9. From the side view of the b-barrel, L8 almost covers one end of the b-barrel.
Three fragments with weak or missing electron densities are all part of the long L8 loop. An analysis of the surface electrostatic potential shows that one side of the molecular ‘head end’ containing Lys-1276, Arg-1280 and Lys-1291 from L8 is highly positively charged (shown in green) while the flanking regions of this end of the b-barrel, formed by L1-b2-L2, L3 and L4 are highly negatively charged (shown in red) as contributed by acidic residues Glu-1170, Asp-1179, Asp-1181, Asp-1183, Asp-1196 and Asp-1220.
An analysis of the closed cavities of the protein reveals that Kgp contains 6 Closed cavities. Most of the cavities contain similar volumes, areas, and radii. The exception is Cavity 4 (shown in yellow), which is roughly half the size of the other cavities.
Ligands and Residues in Kgp:
Within the two chains in the domain, there are several ligands worth mentioning. In Subunit A, there are 3 residues. Although a sulfate is present in the A subunit, it is technically not attached to a binding site, which does not make it a crucial ligand of the protein as compared to the other three ligands.
The ligands in the A subunit include two calcium ligands (Calcium 1 Ligand): In each molecule, a Ca2+ coordinates to Asp-1179, Asp-1181, Asp-1183, Gln-1185, Asn-1221 and one H2O molecule binding L2 tightly to the b-barrel. As there is no Calcium related function known for K2, this ion might act primarily to stabilize the conformation of the loop which surrounds it.
In another Calcium 2 ligand, Calcium coordinates to Thr-1162, Glu-1164, Gly-1202, Asn-1205, Asp-1326 and one H2O molecule interacting with L3 and binding N-terminal b1 to C-terminal b11. The binding of this ion may act to stabilize the overall b-barrel structure of the domain. The one nitrate ligand in the subunit has also been identified; however, the functioned has not been found.
The ligands in the B subunit include two more Calcium residues(Calcium 1 ligand), (Calcium 2 ligand), a Glycerol: One glycerol (added as a cryo-protectant) and one water molecule were found to sit in a closed pocket formed by the fragments of Trp1197–Thr1199, Lys1291–Trp1296 and Tyr1322–Leu1324 only in chain B. With the hydrogen bond connections, these glycerol and water molecules are believed to have stabilized the conformation of fragment Gln1293–Val1295 in chain B. And similar to the A chain, the function of the Nitrate has not been described.
Functionally Important Residues:
Although much is not known about the function of Kgp, 2 sites have been proposed as functionally important with regard to hemolysis. As the Kgp cleavage sites in K2 at Lys-1291 and Lys-1276 are located within loop L8, the data provide evidence that cleavage or truncation specifically affecting this loop critically removes haemolytic capacity. Accordingly, research has shown that proteolytic processing of the loop (at Lys-1291 and perhaps also at Lys-1276) would readily modify the conformational state of L8 leading to loss of capacity to engage in intermolecular associations necessary for haemolytic action (Collyer, 2010).
Isolectric point and Molecular Weight:
The theoretical isoelectric point occurs at a pH of 4.44 and the molecular weight is 38728.46 Da. The isoelectric point signifies the pH at which a the protein carries a zero net electrical charge. The theoretical molecular weight refers to the sum of the atomic weights of all the atoms in a molecule.
Protein Comparison:
The MAM domain of the Receptor-Type Tyrosine-protein Phosphatase (pdb id= 2v5y) is a structure similar to the Lysine gingipain. With a Z score of 14.0, the comparison is significant. Human RPTPmu is a type IIB receptor protein tyrosine phosphatase that both forms an adhesive contact itself and is involved in regulating adhesion by dephosphorylating components of cadherin-catenin complexex (Aricescu et. al, 2007).
Also, Ephrin Receptor Type A (pdb id= 1kgy) is another protein and its chain A is not as structurally comparable as as 2v5y, but contains a significant Z score of 11.7. The Eph family of receptor tyrosine kinases and their membrane-anchored ephrin ligands are important in regulating cell-cell interactions as they initiate a unique bidirectional signal transduction cascade whereby information is communicated into both the Eph-expressing and the ephrin-expressing cells. Ephrins are are also crucia componeceptor tyrosine kinases and their membrane-anchored ephrin ligands are important in regulating cell-cell interactions as they initiate a unique bidirectional signal transduction cascade whereby information is communicated into both the Eph-expressing and the ephrin-expressing cells. Ephrins are are also crucia componentsl promoting higher-order clustering and the initiation of bidirectional signalling (Himanen, 2001). ntsl promoting higher-order clustering and the initiation of bidirectional signalling (Himanen, 2001).
The primary differences between K2 and these homologues are mainly in the loop structures at the ‘head end’ of the b-barrels of the proteins (Collyer, 2010).
Significance:
The lysine gingipain is one of three gingipains in Porphyromonas gingivalis, along with RgpA and RgpB, which are two different Arginine gingipains. However, the structures of all three gingipains are related to a certain degree, with differences being in the side chain (Lysine instead of Arginine) and a few other details that will be studied in greater depth with the Molsoft program. Furthermore, according to Imamura, gingipains are trypsin-like cysteine proteinases and "Kgp is the most potent fibrinogen/fibrin degrading enzume of the 3 gingipains in human plasma and is involved in the bleeding tendency at the diseased gingiva" (2003). Thus, the harmful effects of Kgp need to be addressed, studied in greater detail and cured.
The importance of studying this protein is crucial in the seriousness of P. gingivalis. It is found in the oral cavity, where it is implicated in certain forms of periodontal disease as well as the upper gastrointestinal tract, respiratory tract and in the colon. Additionally Porphyromonas gingivalis has been linked to Rheumatoid Arthritis. Patients with Rheumatoid Arthritis have an increased incidence of periodontal disease and antibodies to the bacterium are significantly more common in patients with Rheumatoid Arthritis (Collyer, 2010).Thus, in researching more about the causes and proteins involved in forming periodontitis, many diseases can be prevented.