Hydrogen/deuterium (H/D) exchange or chemical cross-linking by soluble carbodiimide (EDC) was employed in combination with high-resolution mass spectrometry (MS) to extend our knowledge about contact surface regions involved in the well-characterized model of interaction between two molecules of human 14-3-3ζ regulatory protein. The H/D exchange experiment provided low resolution mapping of interaction in the homodimeric 14-3-3ζ complex. A lower level of deuteration, suggesting structural protection, of two sequential segments has been demonstrated for dimeric 14-3-3ζ wild type relative to the monomeric mutant 14-3-3ζ S58D. The N-terminal sequence (the first 27 residues) from one subunit interacts with region αC'and αD'-helices (residues 45-98) of the other molecule across the dimer interface. To identify interacting amino acid residues within the studied complex, a chemical cross-linking reaction was carried out to produce the covalent homodimer, which was detected by SDS-PAGE. The MS analysis (following tryptic in-gel digestion) employing both high resolution and tandem mass spectrometry revealed cross-linked amino acid residues. Two alternative salt bridges between Glu81 and either Lys9 or the N-terminal amino group have been found to participate in transient interactions of the 14-3-3ζ isotype homodimerization. The data obtained, which have never previously been reported, were used to modify the published 14-3-3 crystal structure using molecular modeling. Based on our findings, utilization of this combination of experimental approaches, which preserve protein native structures, is suitable for mapping the contact between two proteins and also allows for the description of transient interactions or of regions with flexible structure in the studied protein complexes.

• MeSH
• hmotnostní spektrometrie metody   MeSH
• karbodiimidy chemie   MeSH
• konformace proteinů MeSH
• lidé MeSH
• mapování interakce mezi proteiny MeSH
• molekulární sekvence - údaje MeSH
• multimerizace proteinu MeSH
• mutace MeSH
• proteiny 14-3-3 chemie   genetika   izolace a purifikace   metabolismus   MeSH
• reagencia zkříženě vázaná chemie   MeSH
• rekombinantní proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• sekvence aminokyselin MeSH
• simulace molekulární dynamiky MeSH
• vodík-deuteriová výměna metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The host structural maintenance of chromosomes 5/6 complex (Smc5/6) suppresses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing the X protein (HBx), which redirects the cellular DNA damage-binding protein 1 (DDB1)-containing E3 ubiquitin ligase to target Smc5/6 for degradation. However, the details of how HBx modulates the interaction between DDB1 and Smc5/6 remain to be determined. In this study, we performed biophysical analyses of recombinant HBx and functional analysis of HBx mutants in HBV-infected primary human hepatocytes (PHH) to identify key regions and residues that are required for HBx function. We determined that recombinant HBx is soluble and exhibits stoichiometric zinc binding when expressed in the presence of DDB1. Mass spectrometry-based hydrogen-deuterium exchange and cysteine-specific chemical footprinting of the HBx:DDB1 complex identified several HBx cysteine residues (located between amino acids 61 and 137) that are likely involved in zinc binding. These cysteine residues did not form disulfide bonds in HBx expressed in human cells. In line with the biophysical data, functional analysis demonstrated that HBx amino acids 45 to 140 are required for Smc6 degradation and HBV transcription in PHH. Furthermore, site-directed mutagenesis determined that C61, C69, C137, and H139 are necessary for HBx function, although they are likely not essential for DDB1 binding. This CCCH motif is highly conserved in HBV as well as in the X proteins from various mammalian hepadnaviruses. Collectively, our data indicate that the essential HBx cysteine and histidine residues form a zinc-binding motif that is required for HBx function.IMPORTANCE The structural maintenance of chromosomes 5/6 complex (Smc5/6) is a host restriction factor that suppresses HBV transcription. HBV counters this restriction by expressing HBV X protein (HBx), which redirects a host ubiquitin ligase to target Smc5/6 for degradation. Despite this recent advance in understanding HBx function, the key regions and residues of HBx required for Smc5/6 degradation have not been determined. In the present study, we performed biochemical, biophysical, and cell-based analyses of HBx. By doing so, we mapped the minimal functional region of HBx and identified a highly conserved CCCH motif in HBx that is likely responsible for coordinating zinc and is essential for HBx function. We also developed a method to produce soluble recombinant HBx protein that likely adopts a physiologically relevant conformation. Collectively, this study provides new insights into the HBx structure-function relationship and suggests a new approach for structural studies of this enigmatic viral regulatory protein.

• MeSH
• aminokyselinové motivy MeSH
• aminokyseliny MeSH
• DNA vazebné proteiny metabolismus   MeSH
• hepatitida B metabolismus   virologie   MeSH
• interakce hostitele a patogenu MeSH
• lidé MeSH
• rekombinantní fúzní proteiny MeSH
• sekvence aminokyselin MeSH
• trans-aktivátory chemie   metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• virus hepatitidy B fyziologie   MeSH
• zinek metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

AGR2 is an oncogenic endoplasmic reticulum (ER)-resident protein disulfide isomerase. AGR2 protein has a relatively unique property for a chaperone in that it can bind sequence-specifically to a specific peptide motif (TTIYY). A synthetic TTIYY-containing peptide column was used to affinity-purify AGR2 from crude lysates highlighting peptide selectivity in complex mixtures. Hydrogen-deuterium exchange mass spectrometry localized the dominant region in AGR2 that interacts with the TTIYY peptide to within a structural loop from amino acids 131-135 (VDPSL). A peptide binding site consensus of Tx[IL][YF][YF] was developed for AGR2 by measuring its activity against a mutant peptide library. Screening the human proteome for proteins harboring this motif revealed an enrichment in transmembrane proteins and we focused on validating EpCAM as a potential AGR2-interacting protein. AGR2 and EpCAM proteins formed a dose-dependent protein-protein interaction in vitro Proximity ligation assays demonstrated that endogenous AGR2 and EpCAM protein associate in cells. Introducing a single alanine mutation in EpCAM at Tyr251 attenuated its binding to AGR2 in vitro and in cells. Hydrogen-deuterium exchange mass spectrometry was used to identify a stable binding site for AGR2 on EpCAM, adjacent to the TLIYY motif and surrounding EpCAM's detergent binding site. These data define a dominant site on AGR2 that mediates its specific peptide-binding function. EpCAM forms a model client protein for AGR2 to study how an ER-resident chaperone can dock specifically to a peptide motif and regulate the trafficking a protein destined for the secretory pathway.

• MeSH
• adhezní molekula epiteliálních buněk genetika   metabolismus   MeSH
• lidé MeSH
• MFC-7 buňky MeSH
• peptidy metabolismus   MeSH
• proteiny genetika   metabolismus   MeSH
• protoonkogenní proteiny c-mdm2 metabolismus   MeSH
• rekombinantní proteiny metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH