TMC-95's natural cyclic tripeptide metabolites represent potent competitive proteasome inhibitors. The constrained conformation of TMC-95 proteasomal inhibitors provides the driving force for entropically high-affinity binding. Based on the crystal structure of the proteasome:TMC-95A complex, the synthetically challenging TMC-95 core structure was used for the design and synthesis of less demanding biphenyl-ether macrocycles, in which the biphenyl-ether moiety functions as an endocyclic clamp restricting its tripeptide backbone. These simplified analogs allowed us to identify high plasticity of the proteasomal tryptic-like specificity pocket. Biphenyl-ether compounds extended with an amide group were hydrolyzed by the proteasome, although the crystal structure of such proteasome:biphenyl-ether complexes revealed quenching of proteolysis at the acyl-enzyme intermediate. Our data reveal that biphenyl-ether derivatives bind noncovalently to the proteasomal tryptic-like active site in a reversible substrate-like manner without allosteric changes of active site residues.
We have recently developed methods for specific and high-level replacement of methionine with 2-aminohexanoic acid, selenomethionine and telluromethionine as isosteric and atomic analogues for structural investigations of human recombinant annexin V. The variants formed isomorphic crystals and retained the parent three-dimensional fold and bioactivities. Folding parameters were determined from thermal and chemical unfolding to partially denatured states. Stabilities estimated from guanidinium chloride unfolding equilibria are not changed significantly for the atomic mutants (S→Se→Te) while the denaturation midpoint is shifted toward lower values with an increase of the m values at the increase of hydrophobicity. In contrast, stabilities in urea are considerably affected by the atomic substitutions, decreasing together with the m and [D] 1/2 values. The methylene and selenium variants are identical within the limits of error of all measurements performed here. The physical parameters of the amino acid analogues and the values derived from the slopes of the unfolding data are highly correlated. This approach demonstrates how systematic variations in atomic number at the site of replacement (atomic mutations) can provide a method to probe specific folding properties of proteins.Keywords : mutants ; analogues ; folding; methionine; stability.The key to the protein-folding problem lies in the precise attribute altered protein properties to such specific atomic mutations should enable the analyses of distinct contributions of variqualitative and quantitative characterisation of the processes that govern the transition of a particular linear polypeptide into the ous local interactions to the protein fold, such as hydrogen bonding, van der Waals and hydrophobic interactions, thus simplifyunique three-dimensional conformation. As stated by Anfinsen ing the interpretation of the experimental data.[1] the native conformation is determined by the totality of inWe succeeded recently in developing a highly efficient teratomic interactions, and hence by the amino acid sequence, method for the specific and quantitative bioincorporation of the in a given environment. Important experimental tools for folding isosteric Met analogues Ahx, Sem and Tem [2, 3]. This technolstudies are site-directed mutagenesis techniques. But changes in ogy has been applied in the present study to investigate human the primary structure obtained by this experimental approach recombinant annexin V in terms of energetics, stability and foldoften result in controversial or barely interpretable data as seving cooperativity. eral sets of interactions are altered. Thus, it is often difficult to Sequence comparisons usually assign similar hydrophobiciassess whether the observed folding properties are due to the ties to methionine, leucine and isoleucine residues in proteins differences in the shape of the side chain or to the chemical [4, 5], but methionine with its highly flexible side chain differs differences of the atoms involved.significantly from both ...
The complex thermodynamics that govern noncovalent protein-ligand interactions are still not fully understood, despite the exponential increase in experimental structural data available from X-ray crystallography and NMR spectroscopy. The eukaryotic 20S proteasome offers an ideal system for such studies as it contains in duplicate three proteolytically active sites with different substrate specificities. The natural product TMC-95A inhibits these proteolytic centers noncovalently with distinct affinities. X-ray crystallographic analysis of the complexes of the yeast proteasome core particle with this natural inhibitor and two synthetic analogues clearly revealed highly homologous hydrogen-bonding networks involving mainly the peptide backbone despite the strongly differentiated binding affinities to the three active sites of the 20S proteasome. The natural product and the two analogues are constrained in a rigid beta-type extended conformation by the endocyclic biaryl clamp, which preorganizes the peptide backbone for optimal adaptation of the ligands to the active site clefts and thus favors the binding processes entropically. However, the biaryl clamp also dictates the orientation of the P1 and P3 residues and their mode of interaction with the protein binding subsites. This limitation is optimally solved in TMC-95A with the conformationally restricted (Z)-prop-1-enyl group acting as P1 residue, at least for the chymotrypsin-like active site; however, it critically affects the inhibitory potencies of the analogues, thus suggesting the use of less-rigid endocyclic clamps in the design of proteasome inhibitors that allow for a better presentation of residues interacting with the active site clefts of the enzyme.
The C-terminal tetrapeptide amide of gastrin, the shortest sequence of this gastrointestinal hormone capable of exhibiting all the biological properties even though at reduced potency, and the related heptapeptide amide were covalently linked to mono-(6-succinylamino-6-deoxy)-β-cyclodextrin to analyze the effect of the bulky cyclic carbohydrate moiety on recognition of the peptides by the G-protein-coupled CCK−B/gastrin receptor and on their signal transduction potencies. With the four-carbon succinyl spacer and particularly with the additional tripeptide spacer in the heptapeptide/β-cyclodextrin conjugate, full recognition by the receptor was obtained with binding affinities identical to those of the unconjugated tetrapeptide and with a potency comparable to that of full agonists. Docking of this conjugate onto a structure of the human CCK−B receptor derived by homology modeling indicates sufficient free space of the peptide moiety for intermolecular interaction at the putative gastrin binding site, whereby additional interactions of the cyclodextrin with the receptor surface apparently suffice for stabilizing the complex and thus for triggering the full hormonal message. The host/guest complexation of the peptide moiety by the β-cyclodextrin which seems to occur at least in the case of the tetrapeptide conjugate does not suffice in its strength for competing with the receptor recognition. However, multiple presentation of the tetragastrin by its covalent linkage to the heptakis-(6-succinylamino-6-deoxy)-β-cyclodextrin leads to peptide/peptide and/or peptide/cyclodextrin collapses with strong interferences in the receptor recognition process. Retention of full agonism by suitably designed monoconjugates of bioactive peptides with cyclodextrins suggests a highly promising approach for targeting host/guest complexed or covalently bound cytotoxic drugs to specific tumor cells for receptor-mediated internalization.
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