Junji Iwahara

Theory, Practice and Applications of Paramagnetic Relaxation Enhancement for the Characterization of Transient Low-Population States of Biological Macromolecules and Their Complexes

Understanding the function of biological macromolecules and their complexes at the physicochemical level requires knowledge of both their structure and dynamics. Conventional biophysical techniques, such as crystallography and NMR, have yielded incredibly detailed structural information at the atomic level on highly populated static states.1 In the context of the energy landscape representation of macromolecules, highly populated states are located within the global free energy minimum region of a relatively rough free energy hypersurface.2-5 Much less is known, however, about lowly populated, higher free energy states which cannot be * E-mail: G.M.C., mariusc@mail.nih.gov; J.I., j.iwahara@utmb.edu. † National Institutes of Health. ‡ University of Texas Medical Branch. Chem. Rev. 2009, 109, 4108–4139 4108

Visualization of transient encounter complexes in protein–protein association

Kinetic data on a number of protein–protein associations have provided evidence for the initial formation of a pre-equilibrium encounter complex that subsequently relaxes to the final stereospecific complex. Site-directed mutagenesis and brownian dynamics simulations have suggested that the rate of association can be modulated by perturbations in charge distribution outside the direct interaction surfaces. Furthermore, rate enhancement through non-specific binding may occur by either a reduction in dimensionality or the presence of a short-range, non-specific attractive potential. Here, using paramagnetic relaxation enhancement, we directly demonstrate the existence and visualize the distribution of an ensemble of transient, non-specific encounter complexes under equilibrium conditions for a relatively weak protein–protein complex between the amino-terminal domain of enzyme I and the phosphocarrier protein HPr. Neither the stereospecific complex alone nor any single alternative conformation can account fully for the intermolecular paramagnetic relaxation enhancement data. Restrained rigid-body simulated annealing refinement against the paramagnetic relaxation enhancement data enables us to obtain an atomic probability distribution map of the non-specific encounter complex ensemble that qualitatively correlates with the electrostatic surface potentials on the interacting proteins. Qualitatively similar results are presented for two other protein–protein complexes.

Detecting transient intermediates in macromolecular binding by paramagnetic NMR

Macromolecular complex formation is governed by two opposing constraints of specificity and speed. Kinetic and theoretical considerations suggest that significant rate enhancement can be achieved either by reducing the dimensionality of the search process or by the creation of a short-range attractive potential around the target site. This implies the existence of transient intermediates involving non-specific binding modes. Here we show that intermolecular paramagnetic relaxation enhancement (PRE) provides a means of directly detecting the presence of, and investigating the nature of, low population transient intermediates under equilibrium conditions. Applying this approach, we characterize the search process whereby a sequence-specific transcription factor (the homeodomain of HOXD9) binds to non-cognate DNA sites as a means of enhancing the rate of specific association. The PRE data in the fast exchange regime reveal the presence of transient intermediates formed in a stochastic manner at non-cognate sites whose structure is similar to that of the specific complex. Two distinct search processes involving intra- as well as intermolecular translocations can be delineated. The intermolecular PRE method is general and can be readily applied to investigations of transient intermediates in many other macromolecular binding processes.

Structure of sortase, the transpeptidase that anchors proteins to the cell wall of Staphylococcus aureus

Surface proteins of Gram-positive bacteria play important roles during the pathogenesis of human infections and require sortase for anchoring to the cell-wall envelope. Sortase cleaves surface proteins at the LPXTG motif and catalyzes the formation of an amide bond between the carboxyl group of threonine (T) and the amino group of cell-wall crossbridges. The NMR structure of sortase reveals a unique β-barrel structure, in which the active-site sulfhydryl of cysteine-184 is poised for ionization by histidine-120, presumably enabling the resultant thiolate to attack the LPXTG peptide. Calcium binding near the active site stimulates catalysis, possibly by altering the conformation of a surface loop that recognizes newly translocated polypeptides. The structure suggests a mechanistic relationship to the papain/cathepsin proteases and should facilitate the design of new antiinfective agents.

NMR structural and kinetic characterization of a homeodomain diffusing and hopping on nonspecific DNA.

Nonspecific protein–DNA interactions are inherently dynamic and involve both diffusion of the protein along the DNA and hopping of the protein from one DNA molecule or segment to another. Understanding how gene regulatory proteins interact nonspecifically with DNA in terms of both structure and dynamics is challenging because the experimental observables are an ensemble average of many rapidly exchanging states. By using a variety of NMR spectroscopic techniques, including relaxation analysis, paramagnetic relaxation enhancement, and residual dipolar couplings, we have characterized structural and kinetic aspects of the interaction of the HoxD9 homeodomain with a nonspecific, 24-bp DNA duplex in a system in which the protein is not constrained to any particular site. The data reveal that HoxD9 binds to nonspecific DNA with the same binding mode and orientation as that observed in the specific complex. The mobility, however, of Arg side-chains contacting the DNA is increased in the nonspecific complex relative to the specific one. The kinetics of intermolecular translocation between two different nonspecific DNA molecules have also been analyzed and reveal that at high DNA concentrations (such as those present in vivo) direct transfer from one nonspecific complex to another nonspecific DNA molecule occurs without going through the intermediary of free protein. This finding provides a simple mechanism for accelerating the target search in vivo for the specific site in a sea of nonspecific sites by permitting more effective sampling of available DNA sites as the protein jumps from one segment to another.

Asymmetrical roles of zinc fingers in dynamic DNA-scanning process by the inducible transcription factor Egr-1

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 DNA-scanning 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.

Crystal structure of the CENP-B protein-DNA complex: The DNA-binding domains of CENP-B induce kinks in the CENP-B box DNA

The human centromere protein B (CENP‐B), one of the centromere components, specifically binds a 17 bp sequence (the CENP‐B box), which appears in every other α‐satellite repeat. In the present study, the crystal structure of the complex of the DNA‐binding region (129 residues) of CENP‐B and the CENP‐B box DNA has been determined at 2.5 Å resolution. The DNA‐binding region forms two helix–turn–helix domains, which are bound to adjacent major grooves of the DNA. The DNA is kinked at the two recognition helix contact sites, and the DNA region between the kinks is straight. Among the major groove protein‐bound DNAs, this ‘kink–straight–kink’ bend contrasts with ordinary ‘round bends’ (gradual bending between two protein contact sites). The larger kink (43°) is induced by a novel mechanism, ‘phosphate bridging by an arginine‐rich helix’: the recognition helix with an arginine cluster is inserted perpendicularly into the major groove and bridges the groove through direct interactions with the phosphate groups. The overall bending angle is 59°, which may be important for the centromere‐specific chromatin structure.

Dynamics of Lysine Side-Chain Amino Groups in a Protein Studied by Heteronuclear 1H−15N NMR Spectroscopy

Despite their importance in macromolecular interactions and functions, the dynamics of lysine side-chain amino groups in proteins are not well understood. In this study, we have developed the methodology for the investigations of the dynamics of lysine NH3(+) groups by NMR spectroscopy and computation. By using 1H−15N heteronuclear correlation experiments optimized for 15NH3(+) moieties, we have analyzed the dynamic behavior of individual lysine NH3(+) groups in human ubiquitin at 2 °C and pH 5. We modified the theoretical framework developed previously for CH3 groups and used it to analyze 15N relaxation data for the NH3(+) groups. For six lysine NH3(+) groups out of seven in ubiquitin, we have determined model-free order parameters, correlation times for bond rotation, and reorientation of the symmetry axis occurring on a pico- to nanosecond time scale. From CPMG relaxation dispersion experiment for lysine NH3(+) groups, slower dynamics occurring on a millisecond time scale have also been detected for Lys27. The NH3(+) groups of Lys48, which plays a key role as the linkage site in ubiquitination for proteasomal degradation, was found to be highly mobile with the lowest order parameter among the six NH3(+) groups analyzed by NMR. We compared the experimental order parameters for the lysine NH3(+) groups with those from a 1 μs molecular dynamics simulation in explicit solvent and found good agreement between the two. Furthermore, both the computer simulation and the experimental correlation times for the bond rotations of NH3(+) groups suggest that their hydrogen bonding is highly dynamic with a subnanosecond lifetime. This study demonstrates the utility of combining NMR experiment and simulation for an in-depth characterization of the dynamics of these functionally most important side-chains of ubiquitin.

Regulation of Directionality in Bacteriophage λ Site-specific Recombination: Structure of the Xis Protein

Abstract Upon induction of a bacteriophage λ lysogen, a site-specific recombination reaction excises the phage genome from the chromosome of its bacterial host. A critical regulator of this process is the phage-encoded excisionase (Xis) protein, which functions both as a DNA architectural factor and by cooperatively recruiting integrase to an adjacent binding site specifically required for excision. Here we present the three-dimensional structure of Xis and the results of a structure-based mutagenesis study to define the molecular basis of its function. Xis adopts an unusual “winged”-helix motif that is modeled to interact with the major- and minor-grooves of its binding site through a single α-helix and loop structure (“wing”), respectively. The C-terminal tail of Xis, which is required for cooperative binding with integrase, is unstructured in the absence of DNA. We propose that asymmetric bending of DNA by Xis positions its unstructured C-terminal tail for direct contacts with the N-terminal DNA-binding domain of integrase and that an ensuing disordered to ordered transition of the tail may act to stabilize the formation of the tripartite integrase–Xis–DNA complex required for phage excision.

The structure of the Dead ringer–DNA complex reveals how AT‐rich interaction domains (ARIDs) recognize DNA

The AT‐rich interaction domain (ARID) is a DNA‐binding module found in many eukaryotic transcription factors. Using NMR spectroscopy, we have determined the first ever three‐dimensional structure of an ARID–DNA complex (mol. wt 25.7 kDa) formed by Dead ringer from Drosophila melanogaster. ARIDs recognize DNA through a novel mechanism involving major groove immobilization of a large loop that connects the helices of a non‐canonical helix–turn–helix motif, and through a concomitant structural rearrangement that produces stabilizing contacts from a β‐hairpin. Dead ringers preference for AT‐rich DNA originates from three positions within the ARID fold that form energetically significant contacts to an adenine–thymine base step. Amino acids that dictate binding specificity are not highly conserved, suggesting that ARIDs will bind to a range of nucleotide sequences. Extended ARIDs, found in several sequence‐specific transcription factors, are distinguished by the presence of a C‐terminal helix that may increase their intrinsic affinity for DNA. The prevalence of serine amino acids at all specificity determining positions suggests that ARIDs within SWI/SNF‐related complexes will interact with DNA non‐sequence specifically.