Nucleotide-binding cleft

Fig. 1. Ribbon diagrams of Dictyostelium discoideum G-actin (Matsuura et al., 2000) and the Ascaris suum aMSP dimer (Bullock et al., 1996) at the same magnification. Actin consists of four subdomains that surround a nucleotide-binding cleft. The G-actin molecule is asymmetric, so that when it polymerizes, the filament it forms has a characteristic polarity and its two ends differ structurally. By contrast, MSP does not contain a nucleotide binding site and the polymerizing unit is a dimer in which the two MSP molecules are related by twofold rotational symmetry. Polymerization produces filaments composed of two helical subfilaments in which the dimers twofold axes are oriented perpendicular to the helix axis. Consequently, the MSP helices have no polarity and the subfilament ends are identical structurally (Bullock et al., 1998). Reproduced from The Journal of Cell Biology, 2000, vol. 149, pp. 7-12 by copyright permission of the Rockefeller University Press.

The dissociation of bound nucleotides requires an opening of the nucleotide binding cleft. Such movement has been observed for E. coli DnaK and bovine Hsc70 when their respective nucleotide exchange factors, GrpE and Bag-1, are bound. In the absence of such factors, the equilibrium between the closed and the opened states is probably the determining factor for the intrinsic rates of nucleotide dissociation. For... 

The refined structure of the F-actin monomer was found to vary from G-actin primarily in two ways. First, the large and small domains (each of which is comprised of two of the subdomains) adjusted their orientation relative to one another. Second, residues 230-250 in subdomain 4 shifted to close the nucleotide binding cleft. The normal mode refinement approach was therefore able to determine a model for F-actin which derived from low energy distortions to the G-actin structure and minor reorientation of its four subdomains and which fit the diffraction data quite well using a small number of adjustable parameters. 

Gt,a is made up of two domains, a GTPase domain and a helical domain. The GTPase or G-domain indicates that Gt, is a member of the superfamily of regulatory GTPases. In addition, G, possesses a helical domain, which represents a characteristic feature of the heterotrimeric G-proteins. The nucleotide binding site is in a cleft between the two domains. It is assumed that the presence of the helical domain is the reason that bound nucleotide dissociates only very slowly from transducin and that the activated receptor is therefore necessary to initiate the GDP/GTP exchange. 

The structure of the isoleucyl-tRNA synthetase (IleRS) from Thermus ther-mophilus (1045 residues, Mr 120 000) has been solved, as well as its complexes with lie and Val.17 The protein contains a nucleotide binding fold (Chapter 1) that binds ATR The fold has two characteristic ATP binding motifs His-54-Val-55-Gly-56-His-57 and Lys-591-Met-592-Ser-593-Lys-594. In the L-Ile-IleRS complex, a single He is bound at the bottom of the ATP cleft, with the hydrophobic side chain in a hydrophobic pocket, surrounded by Pro-46, Trp-518, and Trp-558. L-Leucine cannot fit into this pocket because of the steric hindrance of one of its terminal methyl groups. Larger amino acids are similarly excluded from this site. In the l-Val-IleRS complex, Val is bound to the same site, but the... 

Glucose and 0-toluoylglucosamine bind to these crystals in the deep cleft that separates the lobes of each subunit (70). ADP or AMP-PNP only bind in the presence of a sugar substrate or inhibitor. Only one nucleotide binds per dimer, and its binding site is located at the point of contact between the two subunits (72). Parts of the binding site are on each subunit. This site has been labeled the I site (72). A schematic drawing of the BII structure with the location of the various binding sites is shown in Fig. 16 (72). 

When compared with post-rigor or pre-powerstroke states the structural effects of cleft closure appear to include the movement of SW1, which opens the nucleotide-binding pocket, together with a twist of the central /Lsheet, which is associated with a large movement of the P-loop that considerably modifies the nucleotide binding site. Partial closure of the actin-binding cleft and a very similar twisting of the /3-sheet were also seen in the nucleotide-free structure of Dictyostelium myosin II reported by Reubold et al. (2003). The myosin V atomic model can be fitted without deformation into the electron microscope three-dimensional (3D) reconstruction of decorated actin (Holmes et al., 2004). For this and other... 

Fig. 4. The actin-binding cleft between the upper (red) and lower (gray) 50K domains (orientation as in Fig. 5A). In A (rigor-like), the cleft is shut. In B (pre-powerstroke), the outer end of the cleft (that forms the actin-binding site) is fully open, but the apex or inner end of the cleft (next to the nucleotide-binding pocket ATP is shown in B) is closed. This closure is brought about by the switch 2 element (SW2) being in the closed conformation. In C (post-rigor), both the outer end and the inner end are open. SW2 is open. In A and B the dispositions of SW2 are similar, but not identical. We refer to them as closed 1 (Cj) and closed 2 (C2), respectively.

The strongly bound pre-powerstroke state or top-of-powerstroke state is the transitory state labeled 4 in Fig. 1. It is experimentally difficult to characterize this either kinetically or structurally. At present, the structure can only be guessed at by an extrapolation of the properties of the adjoining structures. It seems very likely that the actin-binding cleft closes on strong binding in the top-of-powerstroke state. Comparison of the structures of the pre-powerstroke and post-rigor states with the nucleotide-free... 

Surface-exposed parts of the protein have been distinguished by their ability to bind specific antibodies and proteolytic enzymes under nondenaturing conditions (see Figure 16). Many of these epitopes are located at positions in the sequence that correspond to the external side of the suggested nucleotide-binding fold (Mate et al., 1992). Antibodies to fluorescein bind only to denatured FITC-labeled Ca2+-ATPase, and not to the native FITC-labeled enzyme. This is consistent with a location of bound FITC in a hydrophobic cleft corresponding to the ATP site. 

Structurally, each Ga subunit consists of two domains—a GTPase domain, and a a-helical domain. In between these two domains is a cleft where guanine nucleotide binds. Lipid modification of a Cys residue near the amino-terminus of the Ga subunit allows for binding to membrane [8], and the carboxyl terminus of the protein appears important for interaction with receptor. Indeed, the last five residues of Ga are believed to contribute to specificity of interaction [reviewed in 9]. However Ho and Wong [10] have demonstrated that the amino terminus of Gaz is also a critical determinant of its coupling to the delta opioid receptor. 

Figure 34.16. Actin and Hexokinase. A comparison of actin (blue) and hexokinase from yeast (red) reveals structural similarities indicative of homology. Both proteins have a deep cleft in which nucleotides bind. 

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