Tuning halopyridines for covalent protein modification and applying a novel DDAH mutant to quantify methylated arginines

2- and 4-Halopyridines were previously identified as a novel class of protein modifiers. These weakly electrophilic fragment sized molecules were shown to act as quiescent affinity labels that selectively and covalently modify some protein thiols. Covalent modification can occur when a carboxylate side chain is found at a certain distance and orientation from a cysteine thiol. This arrangement of residues catalyzes covalent bond formation by stabilizing the active, protonated form of the pyridine, which allows subsequent attack by the cysteine thiol. This need for catalysis imparts a unique selectivity that sets 2- and 4-halopyridines apart from most electrophilic fragments since efficient modification is not only derived from inherent reactivity and proximity to the target nucleophile, but also by way of a protonation ‘switch’ catalyzed by two residues at the binding site of the target protein. The inherent selectivity of these fragment sized molecules and their relatively straightforward derivitization at multiple positions on the pyridine ring have prompted further study to characterize their unique features as a class of covalent protein modifiers and to explore their utility to engage therapeutic targets. In this work, the features that set 4-halopyridines apart from other fragment-sized electrophiles are identified and characterized. These studies revealed the protonation ‘switch’ allows modulation of halopyridine reactivity through catalysis whereas the non- enzymatic reactivity remains low an effect not obtainable with other electrophilic fragments. After definition of the general requirements for protein modification and identification of methods to alter reactivity, additional protein targets that meet the requirements for 4-halopyridine modification are identified and validated. Modification of one of the identified targets for 4-halopyridines, V-Ki-ras2 Kristen rat sarcoma viral oncogene (KRas), is characterized in detail. It is demonstrated that the 4-halopyridine fragment represents a starting point to selectively modify this historically difficult drug target in cells. In a separate project, enzyme inhibition is studied and applied in a different way. The utility of a substrate-inactivatable Pseudomonas aeruginosa dimethylarginine dimethylaminiohydrolase (PaDDAH) mutant enzyme, PaDDAH T165L, for the quantitative measurement of asymmetric dimethylarginine (ADMA) is evaluated. The catalytic partitioning between normal turnover and self-inactivation enables a low cost quantitative means for measurement of the clinically-relevant biomarker ADMA.Biochemistr

Nanomolar Binding of Peptides Containing Noncanonical Amino Acids by a Synthetic Receptor

This paper describes the molecular recognition of phenylalanine derivatives and their peptides by the synthetic receptor cucurbit[7]uril (Q7). The 4-tert-butyl and 4-aminomethyl derivatives of phenylalanine (tBuPhe and AMPhe) were identified from a screen to have 20–30-fold higher affinity than phenylalanine for Q7. Placement of these residues at the N-terminus of model tripeptides (X-Gly-Gly), resulted in no change in affinity for tBuPhe-Gly-Gly, but a remarkable 500-fold increase in affinity for AMPhe-Gly-Gly, which bound to Q7 with an equilibrium dissociation constant (Kd) value of 0.95 nM in neutral phosphate buffer. Structure–activity studies revealed that three functional groups work in a positively cooperative manner to achieve this extraordinary stability (1) the N-terminal ammonium group, (2) the side chain ammonium group, and (3) the peptide backbone. Addition of the aminomethyl group to Phe substantially improved the selectivity for peptide versus amino acid and for an N-terminal vs nonterminal position. Importantly, Q7 binds to N-terminal AMPhe several orders of magnitude more tightly than any of the canonical amino acid residues. The high affinity, single-site selectivity, and small modification in this system make it attractive for the development of minimal affinity tags

Post-translational maturation of IDA, a peptide signal controlling floral organ abscission in Arabidopsis

The abscission of sepals, petals and stamens in Arabidopsis flowers is controlled by a peptide signal called IDA (Inflorescence Deficient in Abscission). IDA belongs to the large group of small post-translationally modified signaling peptides that are synthesized as larger precursors and require proteolytic processing and specific side chain modifications for signal biogenesis. Using tissue-specific expression of proteinase inhibitors as a novel approach for loss-of-function analysis, we recently identified the peptidases responsible for IDA maturation within the large family of subtilisin-like proteinases (subtilases; SBTs). Further biochemical and physiological assays identified three SBTs (AtSBT5.2, AtSBT4.12, AtSBT4.13) that cleave the IDA precursor to generate the N-terminus of the mature peptide. The C-terminal processing enzyme(s) remain(s) to be identified. While proline hydroxylation was suggested as additional post-translational modification required for IDA maturation, hydroxylated and non-hydroxylated IDA peptides were found to be equally active in bioassays for abscission

Nanomolar Binding of Peptides Containing Noncanonical Amino Acids by a Synthetic Receptor

This paper describes the molecular recognition of phenylalanine derivatives and their peptides by the synthetic receptor cucurbit[7]uril (Q7). The 4-<i>tert</i>-butyl and 4-aminomethyl derivatives of phenylalanine (tBuPhe and AMPhe) were identified from a screen to have 20–30-fold higher affinity than phenylalanine for Q7. Placement of these residues at the N-terminus of model tripeptides (X-Gly-Gly), resulted in no change in affinity for tBuPhe-Gly-Gly, but a remarkable 500-fold increase in affinity for AMPhe-Gly-Gly, which bound to Q7 with an equilibrium dissociation constant (<i>K</i>_d) value of 0.95 nM in neutral phosphate buffer. Structure–activity studies revealed that three functional groups work in a positively cooperative manner to achieve this extraordinary stability (1) the N-terminal ammonium group, (2) the side chain ammonium group, and (3) the peptide backbone. Addition of the aminomethyl group to Phe substantially improved the selectivity for peptide versus amino acid and for an N-terminal vs nonterminal position. Importantly, Q7 binds to N-terminal AMPhe several orders of magnitude more tightly than any of the canonical amino acid residues. The high affinity, single-site selectivity, and small modification in this system make it attractive for the development of minimal affinity tags

Impact of G12 Mutations on the Structure of K‑Ras Probed by Ultraviolet Photodissociation Mass Spectrometry

Single-residue mutations at Gly12 (G12X) in the GTP-ase protein K-Ras can lead to activation of different downstream signaling pathways, depending on the identity of the mutation, through a poorly defined mechanism. Herein, native mass spectrometry combined with top-down ultraviolet photodissociation (UVPD) was employed to investigate the structural changes occurring from G12X mutations of K-Ras. Complexes between K-Ras or the G12X mutants and guanosine 5′-diphosphate (GDP) or GDPnP (a stable GTP analogue) were transferred to the gas phase by nano-electrospray ionization and characterized using UVPD. Variations in the efficiencies of backbone cleavages were observed upon substitution of GDPnP for GDP as well as for the G12X mutants relative to wild-type K-Ras. An increase in the fragmentation efficiency in the segment containing the first 50 residues was observed for the K-Ras/GDPnP complexes relative to the K-Ras/GDP complexes, whereas a decrease in fragmentation efficiency occurred in the segment containing the last 100 residues. Within these general regions, the specific residues at which changes in fragmentation efficiency occurred correspond to the phosphate and guanine binding regions, respectively, and are indicative of a change in the binding motif upon replacement of the ligand (GDP versus GDPnP). Notably, unique changes in UVPD were observed for each G12X mutant with the cysteine and serine mutations exhibiting similar UVPD changes whereas the valine mutation was significantly different. These findings suggest a mechanism that links the identity of the G12X substitution to different downstream effects through long-range conformational or dynamic effects as detected by variations in UVPD fragmentation