Flexible loop

Serpins form very tight complexes with their corresponding serine pro-teinases, thereby inhibiting the latter. A flexible loop region of the serpin binds to the active site of the proteinases. Upon release of the serpin from the complex its polypeptide chain is cleaved by the proteinase in the middle of this loop region and the molecule is subsequently degraded. In addition to the active and cleaved states of the serpins there is also a latent state with an intact polypeptide chain that is functionally inactive and does not bind to the proteinase. 

The setpin fold comprises a compact body of three antiparallel p sheets, A, B and C, which ate partly coveted by a helices (Figure 6.22). In the structure of the uncleaved form of ovalbumin, which can be regarded as the canonical structure of the serpins, sheet A has five strands. The flexible loop starts at the end of strand number 5 of p sheet A (plS in Figure 6.22), then... 

The flexible loop region in the active form of antithrombin (Figure 6.23a) is in the same general position as in ovalbumin but the first few residues form a short sixth p strand in p sheet A inserted between strands pS and pis. Furthermore there is no a helix in the loop which is extended outside the main body of the molecule, ready to be inserted into the active site of thrombin. 

In viw PAI and antithrombin are stabilized in their active forms by binding to vitronectin and heparin, respectively. These two serpins seem to have evolved what Max Perutz has called "a spring-loaded safety catch" mechanism that makes them revert to their latent, stable, inactive form unless the catch is kept in a loaded position by another molecule. Only when the safety catch is in the loaded position is the flexible loop of these serpins exposed and ready for action otherwise it snaps back and is buried inside the protein. This remarkable biological control mechanism is achieved by the flexibility that is inherent in protein structures. 

In the structure of unphosphorylated phosducin that binds to Gpy, Ser 73 points towards the flexible loop of phosducin and not towards Gpy it is, therefore, accessible on the surface for phosphorylation. Phosphorylation of Ser 73 cannot lead to the direct disruption of the phosducin/GpY interaction. Rather, the structure suggests that phosphorylation may lead to conformational changes in the N-terminal domain of phosducin, especially in the flexible loop region, that could weaken or alter the phosducin/GpY interface. 

Although the underlying physics and mathematics used to convert relaxation rates into molecular motions are rather complex (Lipari and Szabo, 1982), the most important parameter obtained from such analyses, the order parameter. S 2, has a simple interpretation. In approximate terms, it corresponds to the fraction of motion experienced by a bond vector that arises from slow rotation as a rigid body of roughly the size of the macromolecule. Thus, in the interior of folded proteins, S2 for Hn bonds is always close to 1.0. In very flexible loops, on the other hand, it may drop as low as 0.6 because subnanosecond motions partially randomize the bond vector before it rotates as a rigid body. 

Fig. 13.1. Cartoon of the aspartyl-tRNA synthetase amino acid binding site. The aspartate ligand is shown, along with the most important recognition residues. Groups that have been mutated in free energy simulations are boxed or circled. Flexible loop and Motif 2 refer to conserved motifs in the enzyme structure...

Limited proteolysis. Flexible regions of proteins can sometimes be removed by digestion of the protein with different proteases. This technique is based on the techniques that were used to determine the core folded regions of proteins, most notably antibodies (Porter, 1973). Limited proteolysis can be used to remove flexible loops of proteins, or separate multidomain proteins into separate domains and has been used successfully in a number of instances (Noel et al., 1993 Sondek et al., 1996 Mazza et al., 2002). 

In the PPA-a-AI 1 complex, a flexible loop of the enzyme, which would normally contact the substrate, is pushed outward to allow entry of the inhibitor, and one of the key aspartate residues is held in a conformation similar to that observed in the free enzyme. Therefore, some changes relative to the carbohydrate complex are required in order to accommodate the inhibitor. The structurally mimetic interactions within the catalytic site are supplemented by other specific protein-protein interactions, with a substantial buried surface area at the interface, involving 50 residues of the enzyme [171]. 

A. Vandemeulebroucke, S. de Vos, E. Van Holsbeke, J. Steyaert, and W. Versees, A flexible loop as a functional element in the catalytic mechanism of nucleoside hydrolase from Trypanosoma vivax, J. Biol. Chem., 283 (2008) 22272-22282. 

Before substrate binding can take place, rubisco must first be activated. This occurs via carbamylation (reaction with CO2) of an essential Lys residue . This promotes the binding of an essential Mg + ion after which the active site is complete. Rubisco can now recognize and bind the first substrate which is ribulose-P2 (D-ribulose 1,5-bisphosphate) . The substrate is bound to the Mg + ion via an inner-sphere coordination of the C2-carbony 1 and C3-hydroxyl groups which appropriately positions and activates the ribulose-P2 for subsequent reaction. Substrate binding causes a flexible loop to close over the active site which buries the active site deep within the protein and restricts access to a small channel just large enough for CO2 (and 02) . 

Inspection of the crystal structure of compound III bound to the active site of HIV PR revealed lipophilic cavities extending off the Sl/ST subsites adjacent to the /-butyl groups of the benzamidine moiety. The cavities are bordered by flexible loops around Pro81/8T and previous crystallographic studies indicated that both loops can move back by up to 2.5 A, extending the size and... 

Schematic representation of the structure of a TIM monomer. Helices and strands are labeled as H and B, respectively. The view is along the axis of the (3 barrel, into the active site. Key catalytic residues Lysl3, His95, and Glul67 are shown along with the helix that binds the substrate phosphate and the flexible loop that covers the substrate during catalysis. Black dots indicate residues in contact with the second monomer of the enzyme. (From Ref.24. Copyright 1991 by Harcourt Brace.)...

The difference in binding affinities for the T- and R-states lies in a flexible loop of residues 280-288, which in the T-state blocks access to the substrate-binding cleft. The universally conserved Asp 238 behaves as a substrate mimic, occupying the P site.137146147 In the R-state this residue moves, allowing P to enter and bind (Fig. 12-5). 

The motion of the flexible loop of precipitated triosephosphate isomerase has been measured by solid-state deuterium NMR in the presence and absence of substrate and transition-state analogs.62 The loop jumps between two conformations at a rate of 3 X 104 s 1 (from the predominant to the less populated form) irrespective of whether a substrate is bound, showing that it is a natural motion of the protein. 

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