The solution structures of free Enzyme I (EI, ∼128 kDa, 575×2 residues), the first enzyme in the bacterial phosphotransferse system and its complex with HPr (∼146 kDa) have been solved using novel methodology that makes use of prior structural knowledge (namely, the structures of the dimeric EIC domain and the isolated EIN domain both free and complexed to HPr), combined with residual dipolar coupling (RDC), small (SAXS) and wide (WAXS) angle X-ray scattering and small angle neutron scattering (SANS) data. The calculational strategy employs conjoined rigid body/torsion/Cartesian simulated annealing, and incorporates improvements in calculating and refining against SAXS/WAXS data that take into account complex molecular shapes in the description of the solvent layer resulting in a better representation of the SAXS/WAXS data. The RDC data orient the symmetrically related EIN domains relative to the C 2 symmetry axis of the EIC dimer, while translational, shape and size information is provided by SAXS/WAXS. The resulting structures are independently validated by SANS. Comparison of the structures of the free EI and the EI-HPr complex with that of the crystal structure of a trapped phosphorylated EI intermediate reveals large (∼70-90°) hinge body rotations of the two subdomains comprising the EIN domain, as well as of the EIN domain relative to the dimeric EIC domain. These large-scale interdomain motions shed light on the structural transitions that accompany the catalytic cycle of EI.The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is a key signal transduction pathway in bacteria whereby active sugar transport across the cell membrane is * Author to whom correspondence should be addressed: G. Marius Clore, Laboratory of Chemical Physics, Building 5, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520. Tel: (301) 496 0782; mariusc@mail.nih.gov. 4 Present address: WCU Biomodulation Major, Department of Agricultural Biotechnology, Seoul National University, 599 Gwanakro, Gwanak-gu, Seoul 151-921, South Korea.Supplementary Information. Sedimentation velocity c(s) distributions; and detailed protocols for docking and simulating annealing refinement. This material is available free of charge via the Internet at http://pubs.acs.org. The atomic coordinates and experimental RDC, SAXS/WAXS and SANS data (accession codes 2KX9 for free EI, and 2XDF for the EI-HPr complex) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public AccessAuthor Manuscript J Am Chem Soc. Author manuscript; available in PMC 2011 September 22. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript coupled to a sequential phosphorylation cascade via a series of bimolecular protein-protein complexes. [1][2][3][4] The initial two steps of the PTS ar...

Egr-1 is an inducible transcription factor that recognizes 9-bp target DNA sites via three zinc finger domains and activates genes in response to cellular stimuli such as synaptic signals and vascular stresses. Using spectroscopic and computational approaches, we have studied structural, dynamic, and kinetic aspects of the DNAscanning process in which Egr-1 is nonspecifically bound to DNA and perpetually changes its location on DNA. Our NMR data indicate that Egr-1 undergoes highly dynamic domain motions when scanning DNA. In particular, the zinc finger 1 (ZF1) of Egr-1 in the nonspecific complex is mainly dissociated from DNA and undergoes collective motions on a nanosecond timescale, whereas zinc fingers 2 and 3 (ZF2 and ZF3, respectively) are bound to DNA. This was totally unexpected because the previous crystallographic studies of the specific complex indicated that all of Egr-1's three zinc fingers are equally involved in binding to a target DNA site. Mutations that are expected to enhance ZF1's interactions with DNA and with ZF2 were found to reduce ZF1's domain motions in the nonspecific complex suggesting that these interactions dictate the dynamic behavior of ZF1. By experiment and computation, we have also investigated kinetics of Egr-1's translocation between two nonspecific DNA duplexes. Our data on the wild type and mutant proteins suggest that the domain dynamics facilitate Egr-1's intersegment transfer that involves transient bridging of two DNA sites. These results shed light on asymmetrical roles of the zinc finger domains for Egr-1 to scan DNA efficiently in the nucleus.NMR spectroscopy | target search process | interdomain dynamics | protein-DNA interactions | simulation I n cellular responses to various stimuli such as signals and stresses, gene regulation by transcription factors is of fundamental importance. Egr-1 (also known as Zif268) is an inducible transcription factor with crucial roles particularly in the brain and cardiovascular systems in mammals. In the brain, Egr-1 is induced by synaptic signals in an activity-dependent manner and activates genes for long-term memory formation and consolidation (1, 2). In the cardiovascular system, Egr-1 is a stress-inducible transcription factor that activates the genes for initiating defense responses against vascular stress and injury (3, 4). Given the short lifetime of induced Egr-1 (typically ∼2 h) (3), rapid gene activation by Egr-1 is important in these biological processes that require an immediate response to the stimuli.The induced Egr-1 protein has to initiate its role by searching for its target DNA sites among billions of DNA base pairs in the nucleus. In the DNA scanning process, transcription factors need to discriminate their target sites from nonspecific sites based on relatively minor differences in DNA structure and sequence. Crystallographic studies demonstrated that Egr-1 recognizes its 9-bp target sequence, GCGTGGGCG, as a monomer via zinc finger domains 1, 2, and 3 (hereafter referred to as ZF1, ZF2, and ZF3) that contact 3 ...

We report direct evidence for deprotonation of a lysine side chain buried in the hydrophobic core of a protein, demonstrating heteronuclear 1H-15N NMR data on the Lys-66 side chain amine (Nzeta) group in the delta-PHS/V66K variant of staphylococcal nuclease. Previous crystallographic study has shown that the Lys-66 Nzeta group is completely buried in the hydrophobic core. On the basis of double and triple resonance experiments, we found that the 1Hzeta and 15Nzeta chemical shifts at pH 8.0 and 6 degrees C for the buried lysine are 0.81 and 23.3 ppm, respectively, which are too abnormal to correspond to the protonated (NH3+) state. Further investigations using a model system suggested that the abnormal 1H and 15N chemical shifts represent the deprotonated (NH2) state of the Lys-66 Nzeta group. More straightforward evidence for the deprotonation was obtained with 2D F1-1H-coupled 1H-15N heteronuclear correlation experiments. Observed 15N multiplets clearly indicated that the spin system for the Lys-66 Nzeta group is AX2 (NH2) rather than AX3 (NH3+). Interestingly, although the amine group is buried in the hydrophobic core, the hydrogen exchange between water and the Lys-66 Nzeta group was found to be relatively rapid (93 s(-1) at -1 degrees C), which suggests the presence of a dynamic process such as local unfolding or water penetration. The partial self-decoupling effect on 15Nzeta multiplets due to the rapid hydrogen exchange is also discussed.