The limited information available on the structure of complexes involving transcription factors and cognate DNA response elements represents a major obstacle in the quest to understand their mechanism of action at the molecular level. We implemented a concerted structural proteomics approach, which combined hydrogen-deuterium exchange (HDX), quantitative protein-protein and protein-nucleic acid cross-linking (XL), and homology analysis, to model the structure of the complex between the full-length DNA binding domain (DBD) of Forkhead box protein O4 (FOXO4) and its DNA binding element (DBE). The results confirmed that FOXO4-DBD assumes the characteristic forkhead topology shared by these types of transcription factors, but its binding mode differs significantly from those of other members of the family. The results showed that the binding interaction stabilized regions that were rather flexible and disordered in the unbound form. Surprisingly, the conformational effects were not limited only to the interface between bound components, but extended also to distal regions that may be essential to recruiting additional factors to the transcription machinery. In addition to providing valuable new insights into the binding mechanism, this project provided an excellent evaluation of the merits of structural proteomics approaches in the investigation of systems that are not directly amenable to traditional high-resolution techniques.

• Keywords
• DNA, FOXO4, cross-linking, molecular modeling, protein, protein-nucleic acid cross-linking, trans-dichlorodiamineplatinum(II), hydrogen-deuterium exchange, transcription factor, transplatin,
• MeSH
• DNA-Binding Proteins chemistry   metabolism   MeSH
• DNA chemistry   metabolism   MeSH
• Mass Spectrometry MeSH
• Molecular Structure MeSH
• Response Elements MeSH
• Transcription Factors chemistry   metabolism   MeSH
• Deuterium Exchange Measurement MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• DNA-Binding Proteins MeSH
• DNA MeSH
• Transcription Factors MeSH

Mouse activating Nkrp1 proteins are commonly described as type II transmembrane receptors with disulfide-linked homodimeric structure. Their function and the manner in which Nkrp1 proteins of mouse strain (C57BL/6) oligomerize are still poorly understood. To assess the oligomerization state of Nkrp1 proteins, mouse activating EGFP-Nkrp1s were expressed in mammalian lymphoid cells and their oligomerization evaluated by Förster resonance energy transfer (FRET). Alternatively, Nkrp1s oligomers were detected by Western blotting to specify the ratio between monomeric and dimeric forms. We also performed structural characterization of recombinant ectodomains of activating Nkrp1 receptors. Nkrp1 isoforms c1, c2 and f were expressed prevalently as homodimers, whereas the Nkrp1a displays larger proportion of monomers on the cell surface. Cysteine-to-serine mutants revealed the importance of all stalk cysteines for protein dimerization in living cells with a major influence of cysteine at position 74 in two Nkrp1 protein isoforms. Our results represent a new insight into the oligomerization of Nkrp1 receptors on lymphoid cells, which will help to determine their function.

• Keywords
• Förster resonance energy transfer, Nkrp1, cysteine, dimerization, disulfide bond arrangement,
• MeSH
• Antigens, Ly analysis   MeSH
• Chlorocebus aethiops MeSH
• COS Cells MeSH
• Jurkat Cells MeSH
• NK Cell Lectin-Like Receptor Subfamily B analysis   chemistry   MeSH
• Humans MeSH
• Protein Multimerization MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Receptors, Immunologic analysis   MeSH
• Protein Refolding MeSH
• Fluorescence Resonance Energy Transfer MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antigens, Ly MeSH
• Klrb1a protein, mouse MeSH Browser
• Klrb1c protein, mouse MeSH Browser
• NK Cell Lectin-Like Receptor Subfamily B MeSH
• Nkrp1f protein, mouse MeSH Browser
• Receptors, Immunologic MeSH

In this article, we present an enhanced sampling method based on a hybrid Hamiltonian which combines experimental distance restraints with a bias dependent from multiple path-dependent variables. This simulation method determines the bias-coordinates on the fly and does not require a priori knowledge about reaction coordinates. The hybrid Hamiltonian accelerates the sampling of proteins, and, combined with experimental distance information, the technique considers the restraints adaptively and in dependency of the system's intrinsic dynamics. We validate the methodology on the dipole relaxation of two water models and the conformational landscape of dialanine. Using experimental NMR-restraint data, we explore the folding landscape of the TrpCage mini-protein and in a second example apply distance restraints from chemical crosslinking/mass spectrometry experiments for the sampling of the conformation space of the Killer Cell Lectin-like Receptor Subfamily B Member 1A (NKR-P1A). The new methodology has the potential to adaptively introduce experimental restraints without affecting the conformational space of the system along an ergodic trajectory. Since only a limited number of input- and no-order parameters are required for the setup of the simulation, the method is broadly applicable and has the potential to be combined with coarse-graining methods.

• Keywords
• enhanced molecular dynamics simulations, protein folding,
• MeSH
• Time Factors MeSH
• Magnetic Resonance Spectroscopy MeSH
• Peptides chemistry   MeSH
• Molecular Dynamics Simulation * MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Peptides MeSH