A Dynamic Protein-Protein Coupling between the TonB-Dependent Transporter FhuA and TonB.

Bacterial outer membrane TonB-dependent transporters function by executing cycles of binding and unbinding to the inner membrane protein TonB. In the vitamin B12 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.

Nucleotide-Independent Copper(II)-Based Distance Measurements in DNA by Pulsed ESR Spectroscopy.

A site-specific Cu2+ binding motif within a DNA duplex for distance measurements by ESR spectroscopy is reported. This motif utilizes a commercially available 2,2'-dipicolylamine (DPA) phosphormadite easily incorporated into any DNA oligonucleotide during initial DNA synthesis. The method only requires the simple post-synthetic addition of Cu2+ without the need for further chemical modification. Notably, the label is positioned within the DNA duplex, as opposed to outside the helical perimeter, for an accurate measurement of duplex distance. A distance of 2.7 nm was measured on a doubly Cu2+ -labeled DNA sequence, which is in exact agreement with the expected distance from both DNA modeling and molecular dynamic simulations. This result suggests that with this labeling strategy the ESR measured distance directly reports on backbone DNA distance, without the need for further modeling. Furthermore, the labeling strategy is structure- and nucleotide-independent.

Structural and dynamic studies of the transcription factor ERG reveal DNA binding is allosterically autoinhibited.

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.

Tunable, mixed-resolution modeling using library-based Monte Carlo and graphics processing units.

Building on our recently introduced library-based Monte Carlo (LBMC) approach, we describe a flexible protocol for mixed coarse-grained (CG)/all-atom (AA) simulation of proteins and ligands. In the present implementation of LBMC, protein side chain configurations are pre-calculated and stored in libraries, while bonded interactions along the backbone are treated explicitly. Because the AA side chain coordinates are maintained at minimal run-time cost, arbitrary sites and interaction terms can be turned on to create mixed-resolution models. For example, an AA region of interest such as a binding site can be coupled to a CG model for the rest of the protein. We have additionally developed a hybrid implementation of the generalized Born/surface area (GBSA) implicit solvent model suitable for mixed-resolution models, which in turn was ported to a graphics processing unit (GPU) for faster calculation. The new software was applied to study two systems: (i) the behavior of spin labels on the B1 domain of protein G (GB1) and (ii) docking of randomly initialized estradiol configurations to the ligand binding domain of the estrogen receptor (ERα). The performance of the GPU version of the code was also benchmarked in a number of additional systems.

Simulating the dynamics and orientations of spin-labeled side chains in a protein-DNA complex.

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 ß-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. C(α)-C(α) 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.