@article{1299975,

title = {Dissecting the binding mode of low affinity phage display peptide ligands to protein targets by hydrogen/deuterium exchange coupled to mass spectrometry},

author = {Ulrike Leurs and Brian Lohse and Shonoi Ming and Philip A Cole and Rasmus P Clausen and Jesper L Kristensen and Kasper D Rand},

doi = {10.1021/ac503137u},

issn = {1520-6882},

year  = {2014},

date = {2014-12-01},

journal = {Anal Chem},

volume = {86},

number = {23},

pages = {11734-41},

abstract = {Phage display (PD) is frequently used to discover peptides capable of binding to biological protein targets. The structural characterization of peptide-protein complexes is often challenging due to their low binding affinities and high structural flexibility. Here, we investigate the use of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to characterize interactions of low affinity peptides with their cognate protein targets. The HDX-MS workflow was optimized to accurately detect low-affinity peptide-protein interactions by use of ion mobility, electron transfer dissociation, nonbinding control peptides, and statistical analysis of replicate data. We show that HDX-MS can identify regions in the two epigenetic regulator proteins KDM4C and KDM1A that are perturbed through weak interactions with PD-identified peptides. Two peptides cause reduced HDX on opposite sides of the active site of KDM4C, indicating distinct binding modes. In contrast, the perturbation site of another PD-selected peptide inhibiting the function of KDM1A maps to a GST-tag. Our results demonstrate that HDX-MS can validate and map weak peptide-protein interactions and pave the way for understanding and optimizing the binding of peptide scaffolds identified through PD and similar ligand discovery approaches.},

keywords = {Binding Sites, Deuterium Exchange Measurement, Histone Demethylases, Humans, Jumonji Domain-Containing Histone Demethylases, Ligands, Mass Spectrometry, Models, Molecular, Molecular Structure, Peptides},

pubstate = {published},

tppubtype = {article}

}

@article{1299980,

title = {Structural basis of nSH2 regulation and lipid binding in PI3Kα},

author = {Michelle S Miller and Oleg Schmidt-Kittler and David M Bolduc and Evan T Brower and Daniele Chaves-Moreira and Marc Allaire and Kenneth W Kinzler and Ian G Jennings and Philip E Thompson and Philip A Cole and L Mario Amzel and Bert Vogelstein and Sandra B Gabelli},

doi = {10.18632/oncotarget.2263},

issn = {1949-2553},

year  = {2014},

date = {2014-07-01},

journal = {Oncotarget},

volume = {5},

number = {14},

pages = {5198-208},

abstract = {We report two crystal structures of the wild-type phosphatidylinositol 3-kinase α (PI3Kα) heterodimer refined to 2.9 Å and 3.4 Å resolution: the first as the free enzyme, the second in complex with the lipid substrate, diC4-PIP$_2$, respectively. The first structure shows key interactions of the N-terminal SH2 domain (nSH2) and iSH2 with the activation loop that suggest a mechanism by which the enzyme is inhibited in its basal state. In the second structure, the lipid substrate binds in a positively charged pocket adjacent to the ATP-binding site, bordered by the P-loop, the activation loop and the iSH2 domain. An additional lipid-binding site was identified at the interface of the ABD, iSH2 and kinase domains. The ability of PI3Kα to bind an additional PIP$_2$ molecule was confirmed in vitro by fluorescence quenching experiments. The crystal structures reveal key differences in the way the nSH2 domain interacts with wild-type p110α and with the oncogenic mutant p110αH1047R. Increased buried surface area and two unique salt-bridges observed only in the wild-type structure suggest tighter inhibition in the wild-type PI3Kα than in the oncogenic mutant. These differences may be partially responsible for the increased basal lipid kinase activity and increased membrane binding of the oncogenic mutant.},

keywords = {Amino Acid Sequence, Animals, Binding Sites, Boron Compounds, Models, Molecular, Molecular Sequence Data, Phosphatidylinositol 3-Kinases, Protein Binding, Protein Conformation, Sf9 Cells, Signal Transduction, Spodoptera, src Homology Domains},

pubstate = {published},

tppubtype = {article}

}

@article{1299939,

title = {Disordered N-Terminal Domain of Human Uracil DNA Glycosylase (hUNG2) Enhances DNA Translocation},

author = {Gaddiel Rodriguez and Alexandre Esadze and Brian P Weiser and Joseph D Schonhoft and Philip A Cole and James T Stivers},

doi = {10.1021/acschembio.7b00521},

issn = {1554-8937},

journal = {ACS Chem Biol},

volume = {12},

number = {9},

pages = {2260-2263},

abstract = {Nuclear human uracil-DNA glycosylase (hUNG2) initiates base excision repair (BER) of genomic uracils generated through misincorporation of dUMP or through deamination of cytosines. Like many human DNA glycosylases, hUNG2 contains an unstructured N-terminal domain that encodes a nuclear localization signal, protein binding motifs, and sites for post-translational modifications. Although the N-terminal domain has minimal effects on DNA binding and uracil excision kinetics, we report that this domain enhances the ability of hUNG2 to translocate on DNA chains as compared to the catalytic domain alone. The enhancement is most pronounced when physiological ion concentrations and macromolecular crowding agents are used. These data suggest that crowded conditions in the human cell nucleus promote the interaction of the N-terminus with duplex DNA during translocation. The increased contact time with the DNA chain likely contributes to the ability of hUNG2 to locate densely spaced uracils that arise during somatic hypermutation and during fluoropyrimidine chemotherapy.},

keywords = {Binding Sites, Biological Transport, DNA, DNA Glycosylases, Humans, Nuclear Localization Signals, Protein Domains},

pubstate = {published},

tppubtype = {article}

}