Mark W. Maciejewski

Discovery and characterization of a family of insecticidal neurotoxins with a rare vicinal disulfide bridge

We have isolated a family of insect-selective neurotoxins from the venom of the Australian funnel-web spider that appear to be good candidates for biopesticide engineering. These peptides, which we have named the Janus-faced atracotoxins (J-ACTXs), each contain 36 or 37 residues, with four disulfide bridges, and they show no homology to any sequences in the protein/DNA databases. The three-dimensional structure of one of these toxins reveals an extremely rare vicinal disulfide bridge that we demonstrate to be critical for insecticidal activity. We propose that J-ACTX comprises an ancestral protein fold that we refer to as the disulfide-directed beta-hairpin.

Solution structure of the single-strand break repair protein XRCC1 N-terminal domain.

XRCC1 functions in the repair of single-strand DNA breaks in mammalian cells and forms a repair complex with β-Pol, ligase III and PARP. Here we describe the NMR solution structure of the XRCC1 N-terminal domain (XRCC1 NTD). The structural core is a β-sandwich with β-strands connected by loops, three helices and two short two-stranded β-sheets at each connection side. We show, for the first time, that the XRCC1 NTD specifically binds single-strand break DNA (gapped and nicked). We also show that the XRCC1 NTD binds a gapped DNA–β-Pol complex. The DNA binding and β-Pol binding surfaces were mapped by NMR and found to be well suited for interaction with single-strand gap DNA containing a 90° bend, and for simultaneously making contacts with the palm-thumb of β-Pol in a ternary complex. The findings suggest a mechanism for preferential binding of the XRCC1 NTD to flexible single-strand break DNA.

Minimotif Miner: a tool for investigating protein function

In addition to large domains, many short motifs mediate functional post-translational modification of proteins as well as protein-protein interactions and protein trafficking functions. We have constructed a motif database comprising 312 unique motifs and a web-based tool for identifying motifs in proteins. Functional motifs predicted by MnM can be ranked by several approaches, and we validated these scores by analyzing thousands of confirmed examples and by confirming prediction of previously unidentified 14-3-3 motifs in EFF-1.

Structure and Mechanism of Action of Sda, an Inhibitor of the Histidine Kinases that Regulate Initiation of Sporulation in Bacillus subtilis

Histidine kinases are used extensively in prokaryotes to monitor and respond to changes in cellular and environmental conditions. In Bacillus subtilis, sporulation-specific gene expression is controlled by a histidine kinase phosphorelay that culminates in phosphorylation of the Spo0A transcription factor. Sda provides a developmental checkpoint by inhibiting this phosphorelay in response to DNA damage and replication defects. We show that Sda acts at the first step in the relay by inhibiting autophosphorylation of the histidine kinase KinA. The structure of Sda, which we determined using NMR, comprises a helical hairpin. A cluster of conserved residues on one face of the hairpin mediates an interaction between Sda and the KinA dimerization/phosphotransfer domain. This interaction stabilizes the KinA dimer, and the two proteins form a stable heterotetramer. The data indicate that Sda forms a molecular barricade that inhibits productive interaction between the catalytic and phosphotransfer domains of KinA.

Solution structure of a dynein motor domain associated light chain.

Dyneins are molecular motors that translocate towards the minus ends of microtubules. In Chlamydomonas flagellar outer arm dynein, light chain 1 (LC1) associates with the nucleotide binding region within the γ heavy chain motor domain and consists of a central leucine-rich repeat section that folds as a cylindrical right handed spiral formed from six β-β-α motifs. This central cylinder is flanked by terminal helical subdomains. The C-terminal helical domain juts out from the cylinder and is adjacent to a hydrophobic surface within the repeat region that is proposed to interact with the dynein heavy chain. The position of the C-terminal domain on LC1 and the unexpected structural similarity between LC1 and U2A′ from the human spliceosome suggest that this domain interacts with the dynein motor domain.

Structural basis for the topological specificity function of MinE.

Correct positioning of the division septum in Escherichia coli depends on the coordinated action of the MinC, MinD and MinE proteins. Topological specificity is conferred on the MinCD division inhibitor by MinE, which counters MinCD activity only in the vicinity of the preferred midcell division site. Here we report the structure of the homodimeric topological specificity domain of Escherichia coli MinE and show that it forms a novel αβ sandwich. Structure-directed mutagenesis of conserved surface residues has enabled us to identify a spatially restricted site on the surface of the protein that is critical for the topological specificity function of MinE.

Minimotif miner 2nd release: a database and web system for motif search

Minimotif Miner (MnM) consists of a minimotif database and a web-based application that enables prediction of motif-based functions in user-supplied protein queries. We have revised MnM by expanding the database more than 10-fold to approximately 5000 motifs and standardized the motif function definitions. The web-application user interface has been redeveloped with new features including improved navigation, screencast-driven help, support for alias names and expanded SNP analysis. A sample analysis of prion shows how MnM 2 can be used. Weblink: http://mnm.engr.uconn.edu, weblink for version 1 is http://sms.engr.uconn.edu.

An automated tool for maximum entropy reconstruction of biomolecular NMR spectra

calculations are computationally costly (that is, efficiency is low). Modern large-scale ∆∆G prediction methods use heuristic algorithms with effective force fields and empirical parameters to estimate the stability changes caused by mutations in agreement with experimental data2–5. There are, however, two considerable drawbacks pertinent to the heuristic methods. First, most of these prediction methods rely on parameter training using available experimental ∆∆G data. Such training is usually biased toward mutations that feature large-to-small residue substitutions, such as alanine-scanning experiments (that is, poor transferability). Second, protein backbone flexibility, which is crucial for resolving atomic clashes and backbone strains in mutant proteins, is not considered in these methods, thereby reducing accuracy and limiting the application of heuristic methods (that is, limited applicability). To address the issues of efficiency, transferability and applicability, we developed the Eris method, which uses a physical force field with atomic modeling as well as fast side-chain packing and backbone relaxation algorithms. The free energy is expressed as a weighted sum of van der Waals forces, solvation, hydrogen bonding and backbone-dependent statistical energies6 (Supplementary Methods online). The weighting parameters are independently trained to recapitulate the native amino acid sequences for 34 proteins using high-resolution X-ray structures6. Additionally, an integral step of Eris is backbone relaxation when severe atom clashes or backbone strains are detected during calculation. We tested Eris on 595 mutants from five proteins, for which the ∆∆G values were documented (Fig. 1a). We found significant agreement between the predicted and measured ∆∆G values with a correlation coefficient of 0.75 (P = 2 × 10−108). The correlation between the predictions and experiments is comparable to that reported using other methods2–5. Unlike previous methods, Eris also has high predictive power for small-to-large3 sidechain-size mutations (Fig. 1b,c), owing to its ability to effectively relax backbone structures and resolve clashes introduced by mutations. As a direct comparison with other methods, we computed the stability changes of the small-to-large mutations using Eris and other web-based stability prediction servers. We found that Eris outperformed other available servers (Supplementary Discussion and Supplementary Tables 1 and 2 online). Additionally, Eris features a protein structure pre-relaxation option, which remarkably improves the prediction accuracy when a highresolution protein structure is not available (Supplementary Discussion and Supplementary Fig. 1 online). Our test validates the unbiased force field, side-chain packing and backbone relaxation algorithms in Eris. We anticipate Eris will be applicable to examining a much larger variety of mutations during protein engineering. We built a web-based Eris server for ∆∆G estimation. The server is freely accessible online (http:// eris.dokhlab.org).

Nonuniform Sampling and Maximum Entropy Reconstruction in Multidimensional NMR

NMR spectroscopy is one of the most powerful and versatile analytic tools available to chemists. The discrete Fourier transform (DFT) played a seminal role in the development of modern NMR, including the multidimensional methods that are essential for characterizing complex biomolecules. However, it suffers from well-known limitations: chiefly the difficulty in obtaining high-resolution spectral estimates from short data records. Because the time required to perform an experiment is proportional to the number of data samples, this problem imposes a sampling burden for multidimensional NMR experiments. At high magnetic field, where spectral dispersion is greatest, the problem becomes particularly acute. Consequently multidimensional NMR experiments that rely on the DFT must either sacrifice resolution in order to be completed in reasonable time or use inordinate amounts of time to achieve the potential resolution afforded by high-field magnets. Maximum entropy (MaxEnt) reconstruction is a non-Fourier method of spectrum analysis that can provide high-resolution spectral estimates from short data records. It can also be used with nonuniformly sampled data sets. Since resolution is substantially determined by the largest evolution time sampled, nonuniform sampling enables high resolution while avoiding the need to uniformly sample at large numbers of evolution times. The Nyquist sampling theorem does not apply to nonuniformly sampled data, and artifacts that occur with the use of nonuniform sampling can be viewed as frequency-aliased signals. Strategies for suppressing nonuniform sampling artifacts include the careful design of the sampling scheme and special methods for computing the spectrum. Researchers now routinely report that they can complete an N-dimensional NMR experiment 3(N-1) times faster (a 3D experiment in one ninth of the time). As a result, high-resolution three- and four-dimensional experiments that were prohibitively time consuming are now practical. Conversely, tailored sampling in the indirect dimensions has led to improved sensitivity. Further advances in nonuniform sampling strategies could enable further reductions in sampling requirements for high resolution NMR spectra, and the combination of these strategies with robust non-Fourier methods of spectrum analysis (such as MaxEnt) represent a profound change in the way researchers conduct multidimensional experiments. The potential benefits will enable more advanced applications of multidimensional NMR spectroscopy to study biological macromolecules, metabolomics, natural products, dynamic systems, and other areas where resolution, sensitivity, or experiment time are limiting. Just as the development of multidimensional NMR methods presaged multidimensional methods in other areas of spectroscopy, we anticipate that nonuniform sampling approaches will find applications in other forms of spectroscopy.

Sparse sampling methods in multidimensional NMR

Although the discrete Fourier transform played an enabling role in the development of modern NMR spectroscopy, it suffers from a well-known difficulty providing high-resolution spectra from short data records. In multidimensional NMR experiments, so-called indirect time dimensions are sampled parametrically, with each instance of evolution times along the indirect dimensions sampled via separate one-dimensional experiments. The time required to conduct multidimensional experiments is directly proportional to the number of indirect evolution times sampled. Despite remarkable advances in resolution with increasing magnetic field strength, multiple dimensions remain essential for resolving individual resonances in NMR spectra of biological macromolecues. Conventional Fourier-based methods of spectrum analysis limit the resolution that can be practically achieved in the indirect dimensions. Nonuniform or sparse data collection strategies, together with suitable non-Fourier methods of spectrum analysis, enable high-resolution multidimensional spectra to be obtained. Although some of these approaches were first employed in NMR more than two decades ago, it is only relatively recently that they have been widely adopted. Here we describe the current practice of sparse sampling methods and prospects for further development of the approach to improve resolution and sensitivity and shorten experiment time in multidimensional NMR. While sparse sampling is particularly promising for multidimensional NMR, the basic principles could apply to other forms of multidimensional spectroscopy.