Characterization of the full-length human Grb7 protein and a phosphorylation representative mutant.

It is well known the dimerization state of receptor tyrosine kinases (RTKs), in conjunction with binding partners such as the growth factor receptor bound protein 7 (Grb7) protein, plays an important role in cell signaling regulation. Previously, we proposed, downstream of RTKs, that the phosphorylation state of Grb7SH2 domain tyrosine residues could control Grb7 dimerization, and dimerization may be an important regulatory step in Grb7 binding to RTKs. In this manner, additional dimerization-dependent regulation could occur downstream of the membrane-bound kinase in RTK-mediated signaling pathways. Extrapolation to the full-length (FL) Grb7 protein, and the ability to test this hypothesis further, has been hampered by the availability of large quantities of pure and stable FL protein. Here, we report the biophysical characterization of the FL Grb7 protein and also a mutant representing a tyrosine-phosphorylated Grb7 protein form. Through size exclusion chromatography and analytical ultracentrifugation, we show the phosphorylated-tyrosine-mimic Y492E-FL-Grb7 protein (Y492E-FL-Grb7) is essentially monomeric at expected physiological concentrations. It has been shown previously the wild-type FL Grb7(WT-FLGrb7) protein is dimeric with a dissociation constant (Kd) of approximately 11µM. Our studies here measure a FL protein dimerization Kd of WT-FL-Grb7 within one order of magnitude at approximately 1µM. The approximate size and shape of the WT-FL-Grb7 in comparison the tyrosine-phosphorylation mimic Y492E-FL-Grb7 protein was determined by dynamic light scattering methods. In vitro phosphorylation of the Grb7SH2 domain indicates only one of the available tyrosine residues is phosphorylated, suggesting the same phosphorylation pattern could be relevant in the FL protein. The biophysical characterization studies in total are interpreted with a view towards understanding the functionally active Grb7 protein conformation.

The run-on oligomer filament enzyme mechanism of SgrAI: Part 1. Assembly kinetics of the run-on oligomer filament.

Filament or run-on oligomer formation by metabolic enzymes is now recognized as a widespread phenomenon having potentially unique enzyme regulatory properties and biological roles, and its dysfunction is implicated in human diseases such as cancer, diabetes, and developmental disorders. SgrAI is a bacterial allosteric type II restriction endonuclease that binds to invading phage DNA, may protect the host DNA from off-target cleavage activity, and forms run-on oligomeric filaments with enhanced DNA-cleavage activity and altered DNA sequence specificity. However, the mechanisms of SgrAI filament growth, cooperativity in filament formation, sequestration of enzyme activity, and advantages over other filament mechanisms remain unknown. In this first of a two-part series, we developed methods and models to derive association and dissociation rate constants of DNA-bound SgrAI in run-on oligomers and addressed the specific questions of cooperativity and filament growth mechanisms. We show that the derived rate constants are consistent with the run-on oligomer sizes determined by EM analysis and are most consistent with a noncooperative growth mode of the run-on oligomer. These models and methods are extended in the accompanying article to include the full DNA-cleavage pathway and address specific questions related to the run-on oligomer mechanism including the sequestration of DNA-cleavage activity and trapping of products.

A transmembrane domain and GxxxG motifs within L2 are essential for papillomavirus infection.

During cellular invasion, human papillomavirus type 16 (HPV16) must transfer its viral genome (vDNA) across the endosomal membrane prior to its accumulation at nuclear PML bodies for the establishment of infection. After cellular uptake, the capsid likely undergoes pH-dependent disassembly within the endo-/lysosomal compartment, thereby exposing hidden domains in L2 that facilitate membrane penetration of L2/vDNA complexes. In an effort to identify regions of L2 that might physically interact with membranes, we have subjected the L2 sequence to multiple transmembrane (TM) domain prediction algorithms. Here, we describe a conserved TM domain within L2 (residues 45 to 67) and investigate its role in HPV16 infection. In vitro, the predicted TM domain adopts an alpha-helical structure in lipid environments and can function as a real TM domain, although not as efficiently as the bona fide TM domain of PDGFR. An L2 double point mutant renders the TM domain nonfunctional and blocks HPV16 infection by preventing endosomal translocation of vDNA. The TM domain contains three highly conserved GxxxG motifs. These motifs can facilitate homotypic and heterotypic interactions between TM helices, activities that may be important for vDNA translocation. Disruption of some of these GxxxG motifs resulted in noninfectious viruses, indicating a critical role in infection. Using a ToxR-based homo-oligomerization assay, we show a propensity for this TM domain to self-associate in a GxxxG-dependent manner. These data suggest an important role for the self-associating L2 TM domain and the conserved GxxxG motifs in the transfer of vDNA across the endo-/lysosomal membrane.

Dimerization in the Grb7 protein.

In previous studies, we showed that the tyrosine phosphorylation state of growth factor receptor-bound protein 7 (Grb7) affects its ability to bind to the transcription regulator FHL2 and the cortactin-interacting protein, human HS-1-associated protein-1. Here, we present results describing the importance of dimerization in the Grb7-Src homology 2 (SH2) domain in terms of its structural integrity and the ability to bind phosphorylated tyrosine peptide ligands. A tyrosine phosphorylation-mimic mutant (Y80E-Grb7-SH2) is largely dimerization deficient and binds a tyrosine-phosphorylated peptide representative of the receptor tyrosine kinase (RTK) erbB2 with differing thermodynamic characteristics than the wild-type SH2 domain. Another dimerization-deficient mutant (F99R-Grb7-SH2) binds the phosphorylated erbB2 peptide with similarly changed thermodynamic characteristics. Both Y80E-Grb7-SH2 and F99R-Grb7-SH2 are structured by circular dichroism measurements but show reduced thermal stability relative to the wild type-Grb7-SH2 domain as measured by circular dichroism and nuclear magnetic resonance. It is well known that the dimerization state of RTKs (as binding partners to adaptor proteins such as Grb7) plays an important role in their regulation. Here, we propose the phosphorylation state of Grb7-SH2 domain tyrosine residues could control Grb7 dimerization, and dimerization may be an important regulatory step in Grb7 binding to RTKs such as erbB2. In this manner, additional dimerization-dependent regulation could occur downstream of the membrane-bound kinase in RTK-mediated signaling pathways.