Structure of the P-Subunit

X.P. Kong, R. Onrust, M. OdonneU, J. Ktuiyan, 3-Dimensional Structure of the p Subunit of Escherichia coli DNA Polymerase-111 Holoenzyme - A Sliding DNA Clamp , Cell, 69,425(1992)... 

Figure 15.6. The P y Subunits of the Heterotrimeric G Protein. Two views illustrate the interaction between the P and the Y subunits. The helices of the y subunit (yellow) wrap around the P subunit (blue). The seven-bladed propeller structure of the P subunit is readily apparent in the representation on the right.

Information about the putative folding of the H,K-ATPase catalytic subunit through the membrane has been obtained by the combined use of hydropathy analysis according to the criteria of Kyte and Doolittle [51], identification of sites sensitive to chemical modification [46,48,50,52-55], and localization of epitopes of monoclonal antibodies [56]. The model of the H,K-ATPase catalytic subunit (Fig. 1) which has emerged from these studies shows ten transmembrane segments and contains cytosolic N- and C-termini [53]. This secondary structure of the catalytic subunit is probably a common feature of the catalytic subunits of P-type ATPases, since evidence supporting a ten a-helical model with cytosolic N- and C-termini has also been published recently for both Ca-ATPase of the sarcoplasmic reticulum and Na,K-ATPase [57-59]. 

Goldberg, J., Huang, H. B., Kwon,Y. G., Greengard, P., Nairn, A. C. and Kuriyan, J. Three-dimensional structure of the catalytic subunit of protein serine/threonine phosphatase-1. Nature 376 745-753,1995. 

It was known that large organic cations such as the tetraethylammonium (TEA) ion and large inactivation peptides (part of the p subunit—T1 assembly) enter the transmembrane pore and the question arises as to how that would take place given the T1 domain s narrow central core. These researchers answer the question in reference 16 through their analysis of the structure and function at the cytoplasmic interface (the connection region between the a and P subunits). 

First, the researchers confirmed that the p subunit s association with the a subunit is disrupted if the T1 domain is removed. The X-ray crystallographic structure of the assembled TI4P4 complex showed why this is the case. The structure shows that large flat surfaces of the p subunit interact with four prominent loops—called contact loops—that extend from the T1 tetramer s... 

However, the latter residue is in no sense equivalent to the Tyr 25 of the P. pantotrophus enzyme. The Tyr 10, which is not an essential residue (19), is provided by the other subunit to that in which it is positioned close to the di heme iron (Fig. 6). In other words, there is a crossing over of the domains. A reduced state structure of the P. aeruginosa enzyme has only been obtained with nitric oxide bound to the d heme iron (20) (Fig. 6). As expected, the heme c domain is unaltered by the reduction, but the Tyr 10 has moved away from the heme d iron, and clearly the hydroxide ligand to the d heme has dissociated so as to allow the binding of the nitric oxide (Fig. 6). This form of the enzyme was prepared by first reducing with ascorbate and then adding nitrite. 

The structure and mechanism of catalysis of FTase were well defined in the late 1990s from several X-ray crystallography and elegant biochemical studies [24,26-30]. The enzyme is a heterodimer of a and P subunits [31,32]. The P subunit contains binding sites for both the farnesyl pyrophosphate and the CAAX protein substrates. A catalytic zinc (Zn " ) identified in the active site of the P subunit participates in the binding and activation of the CAAX protein substrates [28]. The Zn " is coordinated to the enzyme in a distorted tetrahedral geometry and surrounded by hydrophobic pockets [24,27]. Upon binding of the CAAX peptide, the thiol of the cysteine displaces water and is activated for a nucleophilic attack via thiolate on the C-1 carbon atom of farnesyl pyrophosphate [30]. 

Fig. 7.2. Structure and substrate binding sites of Ser/Thr-spedfic protein kinases, a) Peptide binding site structure of the catalytic subunit of the cAMP-dependent protein kinase A from mouse, with bound inhibitor peptide PKI (5-22), shown in dark in the figure. PKI (5-22) is a fragment (amino adds 5-22) of the naturally occurring heat-stable protein kinase inhibitor PKI. The inhibitor peptide binds in the region of the substrate binding site between the two lobes of protein kinase A (Knighton et al., 1991). The P-loop is involved in binding the phosphate residue of ATP. b) ATP binding site structure of casein kinase I with bound Mg-ATP. The Mg is shown as a sphere. MOLSKRIPT representation according to Kraulis, (1991).

Fig. 16.8. Model of inactivation of voltage-gated Na and K channels, a) Inactivation of the Na channel. On inactivation of the Na channel, the loop, which binds domain III and domain IV of the a-subunit, positions itself in the cytoplasmic entrance of the pore and closes it. The indicated hydrophobic amino acids of the connecting loop are involved in the inactivation, b) Inactivation of the K channel. The model assumes that a compact structural part of the C terminus of the P subunit is aligned in the pore and transiently closes it. The inactivating structural part is linked to the pore via a flexible structural element and contains a functionally important leucine residue and a lot of positive charges. According to CatteraU, (1995).

The three-dimensional structure of cholera toxin.6 Side view of the p subunit pentamer as a ribbon drawing. Bound noncovalently to it are five molecules of the ganglioside Gm1 (compare with the structure in Fig. 7-5). The diacyl glycerol parts of the gangliosides are buried in the membrane that lies below the toxin molecule. Courtesy ofW.G.J. Hoi. 

The three-dimensional structure of the GDP complex of the intact transducin heterotrimer195 also shows a tight interaction between a and P subunits. The major interaction is probably disrupted by replacement of the bound GDP by GTP and the conformational change that occurs around the y- phospho group. This explains the dissociation of the a subunit from Py upon activation. An entirely similar picture has been obtained for the action of the inhibitory G protein, Gi2, for which structures of the a subunit and of the aPy heterotrimer (Fig. 11-7,B,C) have been determined.188 233 234 242 The structures resemble those of transducin, but differ in details. 

Kong, X-P, R. Onrust, M. O Donnell, and J. Kuriyan, Three-dimensional structure of the /3 subunit of E. coli DNA polymerase HI holoenzyme A sliding clamp. Cell 69 425-437, 1992. 

These variant Cajfs are clearly members of the P subunit family, but they have quite distinct structural features. For example, the variant 5. mansoni CavP is approximately 25% larger than the conventional schistosome Cavp, and is as much as 50% larger than P subunits from other species. However, the feature of these variant Cajfs that is most striking is found in the BID, where two conserved serine residues that represent consensus protein kinase C (PKC) phosphorylation sites are replaced by other residues (cysteine, alanine). 

M. P. Egloff, P. T. Cohen, P. Reinemer, and D. Barford. Crystal structure of the catalytic subunit of human protein phosphatase 1 and its complex with tungstate. Nature, 376 (6543), 745 753, 1995. 

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