Investigation of GTP-dependent dimerization of G12X K-Ras variants using ultraviolet photodissociation mass spectrometry.

Mutations in the GTPase enzyme K-Ras, specifically at codon G12, remain the most common genetic alterations in human cancers. The mechanisms governing activation of downstream signaling pathways and how they relate back to the identity of the mutation have yet to be completely defined. Here we use native mass spectrometry (MS) combined with ultraviolet photodissociation (UVPD) to investigate the impact of three G12X mutations (G12C, G12V, G12S) on the homodimerization of K-Ras as well as heterodimerization with a downstream effector protein, Raf. Electrospray ionization (ESI) was used to transfer complexes of WT or G12X K-Ras bound to guanosine 5'-diphosphate (GDP) or GppNHp (non-hydrolyzable analogue of GTP) into the gas phase. Relative abundances of homo- or hetero-dimer complexes were estimated from ESI-MS spectra. K-Ras + Raf heterocomplexes were activated with UVPD to probe structural changes responsible for observed differences in the amount of heterocomplex formed for each variant. Holo (ligand-bound) fragment ions resulting from photodissociation suggest the G12X mutants bind Raf along the expected effector binding region (ß-interface) but may interact with Raf via an alternative α-interface as well. Variations in backbone cleavage efficiencies during UV photoactivation of each variant were used to relate mutation identity to structural changes that might impact downstream signaling. Specifically, oncogenic upregulation for hydrogen-bonding amino acid substitutions (G12C, G12S) is achieved by stabilizing ß-interface interactions with Raf, while a bulkier, hydrophobic G12V substitution leads to destabilization of this interface and instead increases the proximity of residues along the α-helical bundles. This study deciphers new pieces of the complex puzzle of how different K-Ras mutations exert influence in downstream signaling.

Selective Covalent Protein Modification by 4-Halopyridines through Catalysis.

We have investigated 4-halopyridines as selective, tunable, and switchable covalent protein modifiers for use in the development of chemical probes. Nonenzymatic reactivity of 4-chloropyridine with amino acids and thiols was ranked with respect to common covalent protein-modifying reagents and found to have reactivity similar to that of acrylamide, but could be switched to a reactivity similar to that of iodoacetamide upon stabilization of the positively charged pyridinium. Diverse, fragment-sized 4-halopyridines inactivated human dimethylarginine dimethylaminohydrolase-1 (DDAH1) through covalent modification of the active site cysteine, acting as quiescent affinity labels that required off-pathway catalysis through stabilization of the protonated pyridinium by a neighboring aspartate residue. A series of 2-fluoromethyl-substituted 4-chloropyridines demonstrated that the pKa and kinact /KI values could be predictably varied over several orders of magnitude. Covalent labeling of proteins in an Escherichia coli lysate was shown to require folded proteins, indicating that alternative proteins can be targeted, and modification is likely to be catalysisdependent. 4-Halopyridines, and quiescent affinity labels in general, represent an attractive strategy to develop reagents with switchable electrophilicity as selective covalent protein modifiers.

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.

Developing an irreversible inhibitor of human DDAH-1, an enzyme upregulated in melanoma.

Inhibitors of the human enzyme dimethylarginine dimethylaminohydrolase-1 (DDAH-1) can raise endogenous levels of asymmetric dimethylarginine (ADMA) and lead to a subsequent inhibition of nitric oxide synthesis. In this study, N(5) -(1-imino-2-chloroethyl)-L-ornithine (Cl-NIO) is shown to be a potent time- and concentration-dependent inhibitor of purified human DDAH-1 (KI =1.3±0.6 µM; kinact =0.34±0.07 min(-1) ), with >500-fold selectivity against two arginine-handling enzymes in the same pathway. An activity probe is used to measure the "in cell" IC50 value (6.6±0.2 µM) for Cl-NIO inhibition of DDAH-1 artificially expressed within cultured HEK293T cells. A screen of diverse melanoma cell lines reveals that a striking 50/64 (78 %) of melanoma lines tested showed increased levels of DDAH-1 relative to normal melanocyte control lines. Treatment of the melanoma A375 cell line with Cl-NIO shows a subsequent decrease in cellular nitric oxide production. Cl-NIO is a promising tool for the study of methylarginine-mediated nitric oxide control and a potential therapeutic lead compound for other indications with elevated nitric oxide production, such as septic shock and idiopathic pulmonary fibrosis.

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 (K(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.