Flap region

NMR is unique in that it can provide detailed and specific information on molecular dynamics in addition to structural information. The use of relaxation time measurements allows the relative mobility of individual atomic positions within a macromolecule to be determined. The d3mamic information obtained includes not only the rates or frequencies of internal motions but also their amplitudes. Such amplitudes are often expressed by order parameters. Not surprisingly, it is observed in many cases that the termini of proteins are more flexible than internal regions. More interestingly, NMR has provided a number of examples where internal loops in proteins have been shown to have dynamics that may be associated with their function. A good example of this is HIV protease, where NMR studies have identified reduced-order parameters in the flap region of the molecule that may reflect flexibility to allow entry of substrates or inhibitors into the active site. 

The protease exists as a homodimer. Each 99-residue monomer contains 10 j3-strands and the dimer is stabilized by a four-stranded antiparallel jS-sheet formed by the N- and C-terminal strands of each monomer. The active site of the enzyme is formed at the interface, where each monomer contributes a catalytic triad (Asp2 -Thr2 -Gly ) that is responsible for cleavage of the protease substrates. The "flap region" is located above the reactive site and is formed by a hairpin from each monomer of two antiparallel j3-strands joined by a j8-turn. There is little difference between the solution and crystal structures of protease-inhibitor complexes, except in those regions where the polypeptide chain is disordered. However, experiments in solution have allowed access to parameters that are not directly accessible from crystal data. These parameters, such as the amplitude and frequency of backbone dynamics, the protonation states of the catalytic aspartate residues, and the rate of monomer interchange, are essential in understanding the interaction of HIV protease with potent inhibitors. 

A widely accepted two-step mechanism of HIV-1 protease binding implies the creation of a loose complex with the open form of the enzyme, followed by the conformational change involving the closure of the flap region over the active site and formation of the final bound complex. Consequently, binding affinity differences between the HIV-1 protease and its mutants may also result from the changes in the internal equilibrium between the bound form of the protease with closed flaps conformation and the unbound open form... 

Fig. 5. The a-carbon positions in the structures of Alcaligenes denitrificans azurin. The cross-hatched circle denotes the Cu atom. A disulfide bridge links Cys 3 and Cys 26. Two important insertions are observed as compared to plastocyanin. The flap region is shown on the right, and an extra loop is at the top of the molecule. (Reproduced with permission from Ref 8.)...

By this point, multiple crystal structures of the enzyme had revealed something unexpected a bound water not seen in mammalian proteases that came between and interacted with two of the carbonyl oxygen atoms of the substrate or inhibitor, and two NHs (from Ile50 and IleSO ) on the flap region of the enzyme. Researchers at DuPont Merck proposed to build in selectivity and take advantage of the entropy released upon displacing this water by... 

Figure 3 is a 3,7 A map of the difference between the electron densities of the pepstatin-bound enzyme and the native enzyme, superimposed on the relevant sections of the electron density map for the native enzyme. It is clear that pepstatin binds in the cleft, and identifies this as the region of active site binding. The density on the difference map corresponding to the pepstatin molecule passes very close to the side chain density of Asp-35. Some of the pepstatin density also lies under the flap region referred to above. 

Figure 8. Proposed general base catalytic mechanism of the acid proteases. "A" shows a schematic drawing of the productive binding mode for substrate RCONHR in the active site of penicillopepsin. The conformational change of the flap region brings Tyr-75 to a position from which it can protonate the amide nitrogen of the scissile bond. Asp-32 attacks the carbon atom of the polarized system through...

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