Sub-competencias de la modelación matemática

The tertiary structures of four M18 aminopeptidases were studied to gain insight on the structure of PfM18AAP and to study the location and conformation of the active site and the spectrin-binding region.

The monomers of the C. acetobutylicum, T. maritima, B. burgdorferi and P.

aeruginosa M18 aminopeptidase crystal structures (Min and Shapiro, 2006) were compared with each other using the Swiss-PdbViewer and showed similar tertiary structures. The monomer of T. maritima was the most complete structure (i.e. no

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gaps were present in the amino acid sequence) and was therefore used for the structure analysis and comparisons with PfM18AAP.

a) b)

c) d)

Figure 54: Crystal structures of the monomers of the M18 aminopeptidases.

Diagram showing similar ribbon structures for the Pseudomonas aeruginosa (2ijz) (a), Clostridium acetobutylicum (2glj) (b), Borrelia burgdorferi (1y7e) (c) and Thermotoga maritima (2glf) (d) M18 aminopeptidases. Ribbon structures were created with DeepView Swiss-PdbViewer, version 3.7 (SP5). The PDB codes of the different proteins are listed in brackets. Blue = domain I and green = domain II.

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The T. maritima aminopeptidase monomer contains two distinct structural regions that will be termed domain I and II (Figure 55a). Domain I is not contiguous and is formed by the N- and C-terminal amino acids of the enzyme. The active site lies at the edge of domain I, in close proximity to domain II, and forms a pocket in which the cofactor-binding amino acids, TmH93, TmD250, TmE280, TmD327 and TmH426 (PfH86, PfD324, PfE380, PfD434 and PfH534) and the two cofactors are located (Figure 55b inset). The two amino acids, TmE279 (PfE379) and TmD95 (PfD88), which are directly and indirectly involved in substrate cleavage are positioned at the edge of the active site. Domain II is formed by T. maritima amino acids Tm97-232 and is thus an internal section of the protein. The histidine, TmH168 (PfH160), that Wilk et al. (2002) found to be essential for enzyme activity in the H. sapiens aspartyl aminopeptidase, is located in an unstructured loop of domain II and faces in the opposite direction to the active site in domain I.

When two monomers (A and B in Figure 55b) associate according to the parameters listed in the crystal structure file (Min and Shapiro, 2006), TmH168 of monomer B completes the active site in monomer A (Figure 55b inset) and vice versa. The histidine, HsH352 (PfH440), speculated to be involved in quaternary structure stabilisation in the H. sapiens aspartyl aminopeptidase (Wilk et al., 2002), is not present in T. maritima and is therefore not shown in the crystal structure. However, if this residue were present, it would be located on domain I of one monomer and could interact with domain II of the other monomer, thereby stabilising the quaternary structure. The putative protein kinase C and casein kinase II phosphorylation sites are located next to a histidine, TmH426 (PfH534), which forms part of the active site in domain I and therefore these two phosphorylation sites could be used to regulate enzymatic activity. The active sites are however located inside the native enzyme (Figure 56) and access to the phosphorylation sites by kinases could be limited. However, phosphorylation could also occur before the monomers are assembled into the quaternary structure.

The T. maritima M18 aminopeptidase occurs as dodecamer in its native state and has the symmetry of a tetrahedron. Because the dimers lie along the edge of the triangles (Figure 56a), three domain Is, each containing an active site, form a

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single vertex and three domain IIs form the flat surface of each triangle in the tetrahedron. Franzetti et al. (2002) described the crystal structure of a tetrahedral M42 aminopeptidase, which is also classified in the MH clan in the MEROPS database. In this enzyme, substrate access to the active sites located within the tetrahedron occurs via pores on the faces (21 Å) and in the vertices (17 Å) of the active enzyme.

Secondary structure prediction showed that the T. maritima aminopeptidase is similar to rPfM18AAP (Figure 47), and therefore the quaternary structure of these two enzymes could be similar. If this is the case, then the amino acids, which form the spectrin-binding region in the PfM18AAP structure, would be located in domain II on the opposite side to domain I (asterisk in Figure 55a). When the transformation parameters of the T. maritima crystal structure file (Min and Shapiro, 2006) are applied to build a dimer and a dodecamer, the spectrin-binding region remains on the surface of these two structures (asterisks in Figure 55b and black amino acids on the surface of the ribbon structure in Figure 56a). If PfM18AAP is a dodecamer, then each triangle face of the tetrahedron would have three spectrin-binding regions extending as loops from its surface (white loops in Figure 56b) and the three active sites would be located on the inside of each vertex of the tetrahedron (red circles in the inset of Figure 56a).

Finally, the putative transmembrane domains of PfM18AAP are found near the C-terminus of the protein. Since the C-C-terminus of the T. maritima aminopeptidase is embedded in domain I (Figure 55) and contributes towards the tertiary structure of the domain (Min and Shapiro, 2006), it is unlikely that the putative transmembrane domains of PfM18AAP function as membrane anchors.

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Figure 55: The T. maritima M18 aminopeptidase monomer and dimer.

Diagram showing the ribbon structure of a monomer (a) and dimer (b) of T.

maritima M18-family aminopeptidase 1 (PDB code: 2glf) (Min and Shapiro, 2006). Domain I of monomer A and B are shown in blue and light blue respectively. Domain II of both monomers are depicted in green and light green respectively. The asterisk marks the predicted location of the PfM18AAP spectrin-binding region in the structure. The inset shows the active site with the two manganese atoms (black dots) surrounded by the metal binding amino acids (red residues: TmH93, TmD250, TmE280, TmD327 and TmH426) and the additional histidine involved in enzyme activity (lavender residue: TmH168). The two amino acids that are directly and indirectly involved in substrate cleavage are located at the edge of the active site (pink residues: TmE279 and TmD95).

Structures were created with DeepView Swiss-PdbViewer, version 3.7 (SP5).

b)

a)

b)

Figure 56: The M18 aminopeptidase dodecamer.

Ribbon structure and diagram showing the quaternary structure of T. maritima M18-family aminopeptidase 1 (PDB code: 2glf) (a) (Min and Shapiro, 2006) and diagram of a possible structure outline of PfM18AAP (b). Dimers (yellow, green and blue) form the edges of the tetrahedron and each triangle face contains three domain IIs (dark blue ribbons in (a) and dark blue circles in (b)), on which the spectrin-binding region of PfM18AAP would be located (black amino acids in (a) or white loops in (b)). Three active sites (red amino acids in (a) and red circles in inset) lie within the vertices of the tetrahedron and the substrate can access these sites via pores located in the faces and vertices of the enzyme. The ribbon structure was created with DeepView Swiss-PdbViewer, version 3.7 (SP5).

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