Sandbox Reserved 1456

The protein, Kgp, is being studied in the bacteria Porphyromonas gingivalis. Kgp is a virulence factor of P. gingivalis that cleaves many constituents of connective tissue, leading to decreased bactericidal activity and chronic inflammation of the gums. Virulence is due to nutrient acquisition, cleavage of host cell surface receptors, signaling via protease activated receptors, and inactivation of cytokines and of the complement system. His444 and Asp388 use acid base catalysis forming an L-lysinylmethyl covalent intermediate with Cys477. Kgp, unlike similar cysteine peptidases, only cleaves after lysine residues and producing a peptide that contains lysine.

Disease

Porphyromonas gingivalis is a Gram-negative oral anaerobe that causes periodontitis. When invaded with Kgp, P. gingivalis degrades the immune and inflammatory response, granting the now-pathogenic bacteria access to the circulatory system. Entry into the circulatory system allows them to cause infection and increase severity of systemic diseases, such as cardiovascular diseases and rheumatoid arthritis ^[1].

Relevance

Bacteria usually benefit human health, but if they are a susceptible host, they can become pathogenic and cause infection and disease. This is happening at a faster rate as the pathogens become more resistant to antibiotics as time passes and the pharmaceutical industry neglects to create new antimicrobials that could combat the growing virulence of these resistant pathogens. By studying Kgp, scientists hope to find a suitable inhibitor for this protein.

Structural highlights

The main present in Kgp are alpha helices and beta sheets, both parallel and anti-parallel. Alpha helices and beta sheets impact how the protein will fold by allowing for specific amino acid interactions. Alpha helices are tightly wound with a center channel too small for even a hydrogen atom to pass through. Alpha helices and beta sheets cannot have a glycine or proline residue as part of the chain and are only found in beta-turns. By knowing this, you know that glycine and proline would not be found in the primary amino acid sequence where the alpha helices and beta sheets would be found.

Kgp is made up of - a catalytic domain (CD) that is split into a smaller subdomain A (orange) and a larger subdomain B (teal) and an immunoglobulin-superfamily domain (IgSF), commonly written as Kgp(CD+IgSF). The subdomain A is representative of the N terminus portion of CD and spans from Asp229-Lys375, and subdomain B is representative of the C terminal domain, spanning form Ser376-Pro600. The rest of the protein is composed of the IgSF, shown in lavender. The two subdomains of CD contribute to tertiary structure integrity because they contain two sodium and two calcium binding sites that stabilize the tertiary structure of Kgp ^[2].

This shows that the atoms that make up Kgp do not leave much room for other molecules to pass through. The primary amino acid sequence determines the quaternary structure of the protein based on the physical and chemical characteristics of the amino acids that make up the protein. This view shows that the primary amino acid sequence folds in such a way that the only molecules that can interfere with the substrate are those that are meant to be there, such as the ligand, CKC, shown in this space-fill view. The binding of the ligand also has to do with the primary amino acid sequence, having chemical properties that work best with those of the ligand to create a stable complex.

The of the surface of Kgp shows a fairly even distribution of hydrophobic (gray) and hydrophilic (purple) atoms. The hydrophilic parts of the protein are accessible to the solvent, as the solvent is usually polar. The hydrophobicity of a surface can give you a quick glance into what the primary structure might look like without going into detail of exactly what amino acids are in that section of the protein.

The of this molecule is (3S)-3,7-diaminoheptan-2-one, referred to as CKC. CKC has the same structure as lysine, having two protonated amine groups and a carboxylic acid group all capable of hydrogen bonding. The side chain of CKC contains four carbon groups which allows for a hydrophobic interaction. This molecule hydrogen bonds with the three amino acids of the catalytic triad to stabilize the structure of Kgp. The structure of the complex is maintained by hydrogen bond formation and a hydrophobic interaction with Trp513, thus decreasing entropy of the system and producing a favorable complex. Image:Catalytic triad.pdf

The of Kgp is made up of Cys477-His444-Asp388. The ligand, CKC, is shown in red and the three amino acids of the catalytic triad are colored by elements (CPK). His444 and Asp388 use acid base catalysis with a covalent intermediate formed with Cys477 to cleave the peptide bond. The histidine imidazolium group transfers a proton to the leaving alpha-amine group of the cleavage product, leaving part of the substrate bound covalently as a thioester to the catalytic Cys477.

The mechanism of action of Kgp is largely determined by its . By binding the ligand to the active site, Kgp is now capable of performing its virulytic activities. The temporary stability of the ligand by the amino acids in the active site allows Kgp to be specific in its activity and achieve optimal function.

There are many other features of the protein that contribute to its overall structure and function. As mentioned previously, Kgp also has two fundamental components, a catalytic domain and an immunoglobulin-superfamily domain. These two aid in the catalytic function of the protein and are connected at ^[3]. Lys601 allows the polypeptide chain to enter the IgSF, essential for folding of Kgp. Without this connection at , Kgp-specific activity would not be possible since proper binding of Kgp and IgSF is absent. In the active site of Kgp, the catalytic triad is formed with Cys477-His444-Asp388. is favored over glutamate: with glutamate protruding slightly more than aspartate, the lysine ligand (CKC) is able to get closer to the catalytic His444, thus allowing the protein to function that much more efficiently ^[4].