G. Marius Clore

The Xplor-NIH NMR molecular structure determination package

We announce the availability of the Xplor-NIH software package for NMR biomolecular structure determination. This package consists of the pre-existing XPLOR program, along with many NMR-specific extensions developed at the NIH. In addition to many features which have been developed over the last 20 years, the Xplor-NIH package contains an interface with a new programmatic framework written in C++. This interface currently supports the general purpose scripting languages Python and TCL, enabling rapid development of new tools, such as new potential energy terms and new optimization methods. Support for these scripting languages also facilitates interaction with existing external programs for structure analysis, structure manipulation, visualization, and spectral analysis.

Determination of three‐dimensional structures of proteins from interproton distance data by hybrid distance geometry‐dynamical simulated annealing calculations

A new hybrid distance space‐real space method for determining three‐dimensional structures of proteins on the basis of interproton distance restraints is presented. It involves the following steps: (i) the approximate polypeptide fold is obtained by generating a set of substructures comprising only a small subset of atoms by projection from multi‐dimensional distance space into three‐dimensional cartesian coordinate space using a procedure known as ‘embedding’; (ii) all remaining atoms are then added by best fitting extended amino acids one residue at a time to the substructures; (iii) the resulting structures are used as the starting point for real space dynamical simulated annealing calculations. The latter involve heating the system to a high temperature followed by slow cooling in order to overcome potential barriers along the pathway towards the global minimum region. This is carried out by solving Newtons equations of motion. Unlike conventional restrained molecular dynamics, however, the non‐bonded interactions are represented by a simple van der Waals repulsion term. The method is illustrated by calculations on crambin (46 residues) and the globular domain of histone H5 (79 residues). It is shown that the hybrid method is more efficient computationally and samples a larger region of conformational space consistent with the experimental data than full metric matrix distance geometry calculations alone, particularly for large systems.

A common sense approach to peak picking in two-, three-, and four-dimensional spectra using automatic computer analysis of contour diagrams☆

The analysis of multidimensional NMR spectra has been a challenging problem since the earliest two-dimensional experiments were reported as stacked plots (I). The first step in analysis involves obtaining a list of chemical-shift coordinates for the cross peaks. Initially, simple programs were used to generate contour plots of twodimensional NMR data, which were analyzed manually. As the utility and versatility of multidimensional NMR spectroscopy grew, several automated methods of peak picking have been developed. This Communication describes a new peak-picking algorithm which is based on contour diagrams and designed for the automated interpretation of higher dimensional 3D and 4D spectra. The oldest and most robust method of analysis is the manual interpretation of 2D contour plots. The strength of manual peak picking results from the relative ease with which the human eye can discriminate real peaks from artifacts and noise. As the proteins studied have become larger, the number of spectra to be analyzed and the number of cross peaks within a spectrum have increased dramatically, with the result that significantly more time and energy are required for the tedious manual peakpicking step. Interactive graphics software, which dynamically maintains a list of peak positions, has to some extent helped with this time-consuming step, particularly with regard to bookkeeping. Although more time will be saved with the automation of peak picking, manual inspection of spectra with an interactive graphics program will always be necessary to verify and edit automated results. Approaches to automated peak picking can be divided into three types: (a) thresholdbased methods; (b) multiplet-symmetry-based methods; and (c) peak-shape-based methods. The simplest automated peak-picking algorithm is based primarily on the intensity of local extrema exceeding a threshold value (2). Uninteresting regions of the spectrum, such as tl noise ridges, are defined to avoid selecting peaks along these artifacts. Since some real peaks have very low intensities, the threshold must be set close to the noise level, which unfortunately results in a very large number of local extrema being picked due to the noise. Thus, by itself the threshold method fails by selecting too many or too few peaks, but is ideally suited as a filter for more sophisticated methods.

Determination of three-dimensional structures of proteins from interproton distance data by dynamical simulated annealing from a random array of atoms Circumventing problems associated with folding

A new real space method, based on the principles of simulated annealing, is presented for determining protein structures on the basis of interproton distance restraints derived from NMR data. The method circumvents the folding problem associated with all real space methods described to date, by starting from a completely random array of atoms and introducing the force constants for the covalent, interproton distance and repulsive van der Waals terms in the target function appropriately. The system is simulated at high temperature by solving Newtons equations of motion. As the values of all force constants are very low during the early stages of the simulation, energy barriers between different folds of the protein can be overcome, and the global minimum of the target function is reliably located. Further, because the atoms are initially only weakly coupled, they can move essentially independently to satisfy the restraints. The method is illustrated using two examples of small proteins, namely crambin (46 residues) and potato carboxypeptidase inhibitor (39 residues).

Molecular basis of human 46X,Y sex reversal revealed from the three-dimensional solution structure of the human SRY-DNA complex.

The solution structure of the specific complex between the high mobility group (HMG) domain of SRY (hSRY-HMG), the protein encoded by the human testis-determining gene, and its DNA target site in the promoter of the müllerian inhibitory substance gene has been determined by multidimensional NMR spectroscopy. hSRY-HMG has a twisted L shape that presents a concave surface (made up of three helices and the N- and C-terminal strands) to the DNA for sequence-specific recognition. Binding of hSRY-HMG to its specific target site occurs exclusively in the minor groove and induces a large conformational change in the DNA. The DNA in the complex has an overall 70 degrees-80 degrees bend and is helically unwound relative to classical A- and B-DNA. The structure of the complex reveals the origin of sequence-specific binding within the HMG-1/HMG-2 family and provides a framework for understanding the effects of point mutations that cause 46X,Y sex reversal at the atomic level.

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.

Solution structure of the N-terminal zinc binding domain of HIV-1 integrase

The solution structure of the N-terminal zinc binding domain (residues 1–55; IN1–55) of HIV-1 integrase has been solved by NMR spectroscopy. IN1–55 is dimeric, and each monomer comprises four helices with the zinc tetrahedrally coordinated to His 12, His 16, Cys 40 and Cys 43. IN1–55 exists in two interconverting conformational states that differ with regard to the coordination of the two histidine side chains to zinc. The different histidine arrangements are associated with large conformational differences in the polypeptide backbone (residues 9–18) around the coordinating histidines. The dimer interface is predominantly hydrophobic and is formed by the packing of the N-terminal end of helix 1, and helices 3 and 4. The monomer fold is remarkably similar to that of a number of helical DMA binding proteins containing a helix-turn-helix (HTH) motif with helices 2 and 3 of IN1–55 corresponding to the HTH motif. In contrast to the DNA binding proteins where the second helix of the HTH motif is employed for DNA recognition, IN1–55 uses this helix for dimerization.

Open-to-closed transition in apo maltose-binding protein observed by paramagnetic NMR.

Large-scale domain rearrangements in proteins have long been recognized to have a critical function in ligand binding and recognition, catalysis and regulation. Crystal structures have provided a static picture of the apo (usually open) and holo usually closed) states. The general question arises as to whether the apo state exists as a single species in which the closed state is energetically inaccessible and interdomain rearrangement is induced by ligand or substrate binding, or whether the predominantly open form already coexists in rapid equilibrium with a minor closed species. The maltose-binding protein (MBP), a member of the bacterial periplasmic binding protein family, provides a model system for investigating this problem because it has been the subject of extensive studies by crystallography, NMR and other biophysical techniques. Here we show that although paramagnetic relaxation enhancement (PRE) data for the sugar-bound form are consistent with the crystal structure of holo MBP, the PRE data for the apo state are indicative of a rapidly exchanging mixture (ns to μs regime) of a predominantly (∼95%) open form (represented by the apo crystal structure) and a minor (∼5%) partially closed species. Using ensemble simulated annealing refinement against the PRE data we are able to determine a 〈r-6〉 ensemble average structure of the minor apo species and show that it is distinct from the sugar-bound state.

The solution structure of an HMG-I(Y)-DNA complex defines a new architectural minor groove binding motif.

The solution structure of a complex between a truncated form of HMG-I(Y), consisting of the second and third DNA binding domains (residues 51–90), and a DNA dodecamer containing the PRDII site of the interferon-β promoter has been solved by multidimensional nuclear magnetic resonance spectroscopy. The stoichiometry of the complex is one molecule of HMG-I(Y) to two molecules of DNA. The structure reveals a new architectural minor groove binding motif which stabilizes B-DNA, thereby facilitating the binding of other transcription factors in the opposing major groove. The interactions involve a central Arg-Gly-Arg motif together with two other modules that participate in extensive hydrophobic and polar contacts. The absence of one of these modules in the third DNA binding domain accounts for its ∼100 fold reduced affinity relative to the second one.