The Ets-Related Gene (ERG) belongs to the Ets family of transcription factors and is critically important for maintenance of the hematopoietic stem cell population. A chromosomal translocation observed in the majority of human prostate cancers leads to the aberrant overexpression of ERG. We have identified regions flanking the ERG Ets domain responsible for autoinhibition of DNA binding and solved crystal structures of uninhibited, autoinhibited, and DNA-bound ERG. NMR-based measurements of backbone dynamics show that uninhibited ERG undergoes substantial dynamics on the millisecond-to-microsecond timescale but autoinhibited and DNA-bound ERG do not. We propose a mechanism whereby the allosteric basis of ERG autoinhibition is mediated predominantly by the regulation of Ets-domain dynamics with only modest structural changes.

Site-directed spin labeling, wherein a nitroxide side chain is introduced into a protein at a selected mutant site, is increasingly employed to investigate biological systems by electron spin resonance (ESR) spectroscopy. An understanding of the packing and dynamics of the spin label is needed to extract the biologically relevant information about the macromolecule from ESR measurements. In this work, molecular dynamics (MD) simulations were performed on the spin labeled restriction endonuclease, EcoRI in complex with DNA. Mutants of this homodimeric enzyme were previously constructed and distance measurements were performed using the Double Electron Electron Resonance experiment. These correlated distance constraints have been leveraged with MD simulations to learn about side chain packing and preferred conformers of the spin label on sites in an α-helix and a β-strand. We found three dihedral angles of the spin label side chain to be most sensitive to the secondary structure where the spin label was located. Conformers sampled by the spin label differed between secondary structures as well. Cα-Cα distance distributions were constructed and used to extract details about the protein backbone mobility at the two spin labeled sites. These simulation studies enhance our understanding of the behavior of spin labels in proteins and thus expand the ability of ESR spectroscopy to contribute to knowledge of protein structure and dynamics.

Bacterial outer membrane TonB-dependent transporters function by executing cycles of binding and unbinding to the inner membrane protein TonB. In the vitamin B transporter BtuB and the ferric citrate transporter FecA, substrate binding increases the periplasmic exposure of the Ton box, an energy-coupling segment. This increased exposure appears to enhance the affinity of the transporter for TonB. Here, continuous wave and pulse EPR spectroscopy were used to examine the state of the Ton box in the Escherichia coli ferrichrome transporter FhuA. In its apo state, the Ton box of FhuA samples a broad range of positions and multiple conformational substates. When bound to ferrichrome, the Ton box does not extend further into the periplasm, although the structural states sampled by the FhuA Ton box are altered. When bound to a soluble fragment of TonB, the TonB-FhuA complex remains heterogeneous and dynamic, indicating that TonB does not make strong, specific contacts with either the FhuA barrel or the core region of the transporter. This result differs from that seen in the crystal structure of the TonB-FhuA complex. These data indicate that unlike BtuB and FecA, the periplasmic exposure of the Ton box in FhuA does not change significantly in the presence of substrate and that allosteric control of transporter-TonB interactions functions by a different mechanism than that seen in either BtuB or FecA. Moreover, the data indicate that models involving a rotation of TonB relative to the transporter are unlikely to underlie the mechanism that drives TonB-dependent transport.

KeywordsEPR spectroscopy; DEER; DNA binding protein; structure elucidation; protein flexibility; noncognate complexIn this communication, we show that the EcoRI restriction endonuclease binds different classes of DNA sites in the same binding cleft. EcoRI generates widespread interest because it exhibits an extraordinary sequence selectivity to carry out its function of cleaving incoming foreign DNA without causing potentially lethal cleavage of cellular DNA. For example, EcoRI binds to its correct recognition site GAATTC up to 90,000-fold better than to miscognate sites that have one incorrect base pair. [1,2] The ∼650 specific sites in the E. coli genome are protected from cleavage by double-strand methylation. The ∼21,000 miscognate sites are not methylated, but are still cleaved by EcoRI with a second-order rate constant that is ∼10 9 -fold lower.[1,2] EcoRI forms only non-specific complexes with no cleavage at sites that differ from GAATTC by two or more base pairs. [1,2] In order to understand the source of such high specificity, it is necessary to determine how the structures of EcoRI complexes differ at specific, miscognate (5/6 bp match), and nonspecific (≤4/6 bp match) DNA sites. This effort is timely given the extensive genetic, biochemical and biophysical data on EcoRI.[1-9] Footprinting results [1] suggest that the three classes of complexes are "structurally" distinct, and thermodynamic profiles (ΔG°, ΔH°, ΔS°, ΔC°P) [3,4] suggest that the specific complex has more restricted conformationalvibrational mobility of the protein and DNA. There are crystal structures of the free protein, Figure 1 shows the structure of the EcoRI specific complex. [6,7] The protein contains a large, relatively rigid and structured globular "main" domain and a smaller "arm" region. The protein arms are invisible in the free protein[6] but become ordered and enfold the DNA in the specific complex, where they play a role in modulating specificity.[2,4] Mutations R131C, S180C, and K249C-S180C were chosen based on the crystal structure. [6,7] These sites are solvent accessible and therefore likely to spin label with minimal perturbation to protein structure. Residues R131 and S180 lie in the inner and outer arms, respectively. Residue K249 is in the main domain, which has very restricted movement [6] and acts as a reference point. Since EcoRI is a 62 kDa homodimer, single cysteine mutations provide two sites for spin labeling, and double mutations provide four sites.The proteins were spin labeled at the cysteines with the methanethiosulfonate spin label (MTSSL). There is an intrinsic cysteine at position 218, but it is buried, leading to <10% labeling even with a 100-fold molar excess of the spin label. The mutant proteins and their spin labeled derivatives catalyze DNA cleavage and have DNA binding affinities similar to that of wild type EcoRI, indicating that they are functionally active (Supporting Information).DEER experiments [11] were performed on spin labeled S180C specific and non-specific complexes, and on R131C and...