Stereochemical control of ribosomal peptidyltransferase reaction. Role of amino acid side-chain orientation of acceptor substrate

AbstractFibrin polymerizes via noncovalent and dynamic association of thrombin-exposed “knobs” with complementary “holes.” Synthetic knob peptides have received significant interest as a means for understanding fibrin assembly mechanisms and inhibiting fibrin polymerization. Nevertheless, the inability to crystallize short peptides significantly limits our understanding of knob peptide structural features that regulate dynamic knob:hole interactions. In this study, we used molecular simulations to generate the first predicted structure(s) of synthetic knobs in solution before fibrin hole engagement. Combining surface plasmon resonance (SPR), we explored the role of structural and electrostatic properties of knob “A” mimics in regulating knob:hole binding kinetics. SPR results showed that association rates were most profoundly affected by the presence of both additional prolines as well as charged residues in the sixth to seventh positions. Importantly, analyzing the structural dynamics of the peptides through simulation indicated that the 3Arg side chain orientation and peptide backbone stability each contribute significantly to functional binding. These findings provide insights into early fibrin protofibril assembly dynamics as well as establishing essential design parameters for high-affinity knob mimics that more efficiently compete for hole occupancy, parameters realized here through a novel knob mimic displaying a 10-fold higher association rate than current mimics.

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Interaction Profiles of Amino Acid Side-Chain Analog Pairs within Membrane Environments

Biophysical Journal ◽

10.1016/j.bpj.2013.11.2791 ◽

2014 ◽

Vol 106 (2) ◽

pp. 499a

Author(s):

Vahid Mirjalili ◽

Michael Feig

Keyword(s):

Amino Acid ◽

Side Chain ◽

Amino Acid Side Chain

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The Role of Arg13 in Protein Phosphatase M tPphA from Thermosynechococcus elongatus

Enzyme Research ◽

10.1155/2012/272706 ◽

2012 ◽

Vol 2012 ◽

pp. 1-7 ◽

Cited By ~ 1

Author(s):

Jiyong Su ◽

Karl Forchhammer

Keyword(s):

Amino Acid ◽

Protein Phosphatase ◽

Arginine Residue ◽

Side Chain ◽

Wild Type ◽

Catalytic Center ◽

Nitrophenyl Phosphate ◽

Reduced Activity ◽

Amino Acid Variants

A highly conserved arginine residue is close to the catalytic center of PPM/PP2C-type protein phosphatases. Different crystal structures of PPM/PP2C homologues revealed that the guanidinium side chain of this arginine residue can adopt variable conformations and may bind ligands, suggesting an important role of this residue during catalysis. In this paper, we randomly mutated Arginine 13 of tPphA, a PPM/PP2C-type phosphatase from Thermosynechococcus elongatus, and obtained 18 different amino acid variants. The generated variants were tested towards p-nitrophenyl phosphate and various phosphopeptides. Towards p-nitrophenyl phosphate as substrate, twelve variants showed 3–7 times higher Km values than wild-type tPphA and four variants (R13D, R13F, R13L, and R13W) completely lost activity. Strikingly, these variants were still able to dephosphorylate phosphopeptides, although with strongly reduced activity. The specific inability of some Arg-13 variants to hydrolyze p-nitrophenyl phosphate highlights the importance of additional substrate interactions apart from the substrate phosphate for catalysis. The properties of the R13 variants indicate that this residue assists in substrate binding.

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