Torsten Herrmann

Protein NMR Structure Determination with Automated NOE Assignment Using the New Software CANDID and the Torsion Angle Dynamics Algorithm DYANA

Combined automated NOE assignment and structure determination module (CANDID) is a new software for efficient NMR structure determination of proteins by automated assignment of the NOESY spectra. CANDID uses an iterative approach with multiple cycles of NOE cross-peak assignment and protein structure calculation using the fast DYANA torsion angle dynamics algorithm, so that the result from each CANDID cycle consists of exhaustive, possibly ambiguous NOE cross-peak assignments in all available spectra and a three-dimensional protein structure represented by a bundle of conformers. The input for the first CANDID cycle consists of the amino acid sequence, the chemical shift list from the sequence-specific resonance assignment, and listings of the cross-peak positions and volumes in one or several two, three or four-dimensional NOESY spectra. The input for the second and subsequent CANDID cycles contains the three-dimensional protein structure from the previous cycle, in addition to the complete input used for the first cycle. CANDID includes two new elements that make it robust with respect to the presence of artifacts in the input data, i.e. network-anchoring and constraint-combination, which have a key role in de novo protein structure determinations for the successful generation of the correct polypeptide fold by the first CANDID cycle. Network-anchoring makes use of the fact that any network of correct NOE cross-peak assignments forms a self-consistent set; the initial, chemical shift-based assignments for each individual NOE cross-peak are therefore weighted by the extent to which they can be embedded into the network formed by all other NOE cross-peak assignments. Constraint-combination reduces the deleterious impact of artifact NOE upper distance constraints in the input for a protein structure calculation by combining the assignments for two or several peaks into a single upper limit distance constraint, which lowers the probability that the presence of an artifact peak will influence the outcome of the structure calculation. CANDID test calculations were performed with NMR data sets of four proteins for which high-quality structures had previously been solved by interactive protocols, and they yielded comparable results to these reference structure determinations with regard to both the residual constraint violations, and the precision and accuracy of the atomic coordinates. The CANDID approach has further been validated by de novo NMR structure determinations of four additional proteins. The experience gained in these calculations shows that once nearly complete sequence-specific resonance assignments are available, the automated CANDID approach results in greatly enhanced efficiency of the NOESY spectral analysis. The fact that the correct fold is obtained in cycle 1 of a de novo structure calculation is the single most important advance achieved with CANDID, when compared with previously proposed automated NOESY assignment methods that do not use network-anchoring and constraint-combination.

A new type V toxin-antitoxin system where mRNA for toxin GhoT is cleaved by antitoxin GhoS.

SUMMARY Among bacterial toxin/antitoxin (TA) systems, to date no antitoxin has been identified that functions by cleaving toxin mRNA. Here we demonstrate YjdO (renamed GhoT) is a membrane lytic peptide that causes ghost cell formation (lysed cells with damaged membranes) and increases persistence (persister cells are tolerant to antibiotics without undergoing genetic change). GhoT is part of a novel TA system with YjdK (renamed GhoS) since in vitro RNA degradation studies, qRT-PCR, and whole-transcriptome studies revealed GhoS masks GhoT toxicity by cleaving specifically ghoT mRNA. Alanine substitutions showed arginine 28 is important for GhoS activity, and RNA sequencing indicated the GhoS cleavage site is rich in uridine and adenosine. The NMR structure of GhoS indicates it is related to the CAS2 CRISPR RNase, and GhoS is a monomer. Hence, GhoT/GhoS is the first type V TA system where a protein antitoxin inhibits the toxin by cleaving specifically its mRNA.

NMR structure of the calreticulin P-domain

The NMR structure of the rat calreticulin P-domain, comprising residues 189–288, CRT(189–288), shows a hairpin fold that involves the entire polypeptide chain, has the two chain ends in close spatial proximity, and does not fold back on itself. This globally extended structure is stabilized by three antiparallel β-sheets, with the β-strands comprising the residues 189–192 and 276–279, 206–209 and 262–265, and 223–226 and 248–251, respectively. The hairpin loop of residues 227–247 and the two connecting regions between the β-sheets contain a hydrophobic cluster, where each of the three clusters includes two highly conserved tryptophyl residues, one from each strand of the hairpin. The three β-sheets and the three hydrophobic clusters form a repeating pattern of interactions across the hairpin that reflects the periodicity of the amino acid sequence, which consists of three 17-residue repeats followed by three 14-residue repeats. Within the global hairpin fold there are two well-ordered subdomains comprising the residues 219–258, and 189–209 and 262–284, respectively. These are separated by a poorly ordered linker region, so that the relative orientation of the two subdomains cannot be precisely described. The structure type observed for CRT(189–288) provides an additional basis for functional studies of the abundant endoplasmic reticulum chaperone calreticulin.

Protein Structure Determination from 13C Spin-Diffusion Solid-State NMR Spectroscopy

Proton-driven 13C spin diffusion (PDSD) is a simple and robust two-dimensional NMR experiment. It leads to spectra with a high signal-to-noise ratio in which cross-peaks contain information about internuclear distances. We show that the total information content is sufficient to determine the atomic-resolution structure of a small protein from a single, uniformly 13C-, 15N-labeled microcrystalline sample. For the example of ubiquitin, the structure was determined by a manual procedure followed by an automatic optimization of the manual structure as well as by a fully automated structure determination approach. The relationship between internuclear distances and cross-peak intensities in the spectra is investigated.

Fast Resonance Assignment and Fold Determination of Human Superoxide Dismutase by High-Resolution Proton-Detected Solid-State MAS NMR Spectroscopy

Re-protonation is key: A combination of a high magnetic field (1 GHz) and ultra-fast magic-angle spinning (60 kHz) allows easy detection of NMR spectra revealing details of secondary and tertiary structures of medium-sized proteins. The technique was applied to the 153-residue microcrystalline Zn II-loaded superoxide dismutase (ZnII-SOD) fully [ 2H,13C,15N]-labeled and 100% re-protonated at the exchangeable sites. Copyright

Structure and backbone dynamics of a microcrystalline metalloprotein by solid-state NMR

We introduce a new approach to improve structural and dynamical determination of large metalloproteins using solid-state nuclear magnetic resonance (NMR) with 1H detection under ultrafast magic angle spinning (MAS). The approach is based on the rapid and sensitive acquisition of an extensive set of 15N and 13C nuclear relaxation rates. The system on which we demonstrate these methods is the enzyme Cu, Zn superoxide dismutase (SOD), which coordinates a Cu ion available either in Cu+ (diamagnetic) or Cu2+ (paramagnetic) form. Paramagnetic relaxation enhancements are obtained from the difference in rates measured in the two forms and are employed as structural constraints for the determination of the protein structure. When added to 1H-1H distance restraints, they are shown to yield a twofold improvement of the precision of the structure. Site-specific order parameters and timescales of motion are obtained by a Gaussian axial fluctuation (GAF) analysis of the relaxation rates of the diamagnetic molecule, and interpreted in relation to backbone structure and metal binding. Timescales for motion are found to be in the range of the overall correlation time in solution, where internal motions characterized here would not be observable.

Rapid Proton-Detected NMR Assignment for Proteins with Fast Magic Angle Spinning

Using a set of six 1H-detected triple-resonance NMR experiments, we establish a method for sequence-specific backbone resonance assignment of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of 5–30 kDa proteins. The approach relies on perdeuteration, amide 2H/1H exchange, high magnetic fields, and high-spinning frequencies (ωr/2π ≥ 60 kHz) and yields high-quality NMR data, enabling the use of automated analysis. The method is validated with five examples of proteins in different condensed states, including two microcrystalline proteins, a sedimented virus capsid, and two membrane-embedded systems. In comparison to contemporary 13C/15N-based methods, this approach facilitates and accelerates the MAS NMR assignment process, shortening the spectral acquisition times and enabling the use of unsupervised state-of-the-art computational data analysis protocols originally developed for solution NMR.

NMR Structure and Metal Interactions of the CopZ Copper Chaperone

A recently discovered family of proteins that function as copper chaperones route copper to proteins that either require it for their function or are involved in its transport. InEnterococcus hirae the copper chaperone function is performed by the 8-kDa protein CopZ. This paper describes the NMR structure of apo-CopZ, obtained using uniformly15N-labeled CopZ overexpressed in Escherichia coli and NMR studies of the impact of Cu(I) binding on the CopZ structure. The protein has a βαββαβ fold, where the four β-strands form an antiparallel twisted β-sheet, and the two helices are located on the same side of the β-sheet. A sequence motif GMXCXXC in the loop between the first β-strand and the first α-helix contains the primary ligands, which bind copper(I). Binding of copper(I) caused major structural changes in this molecular region, as manifested by the fact that most NMR signals of the loop and the N-terminal part of the first helix were broadened beyond detection. This effect was strictly localized, because the remainder of the apo-CopZ structure was maintained after addition of Cu(I). NMR relaxation data showed a decreased correlation time of overall molecular tumbling for Cu(I)-CopZ when compared withapo-CopZ, indicating aggregation of Cu(I)-CopZ. The structure of CopZ is the first three-dimensional structure of a cupro-protein for which the metal ion is an exchangeable substrate rather than an integral part of the structure. Implications of the present structural work for the in vivo function of CopZ are discussed, whereby it is of special interest that the distribution of charged residues on the CopZ surface is highly uneven and suggests preferred recognition sites for other proteins that might be involved in copper transfer.

Structural basis of chaperone-subunit complex recognition by the type 1 pilus assembly platform FimD

Adhesive type 1 pili from uropathogenic Escherichia coli are filamentous protein complexes that are attached to the assembly platform FimD in the outer membrane. During pilus assembly, FimD binds complexes between the chaperone FimC and type 1 pilus subunits in the periplasm and mediates subunit translocation to the cell surface. Here we report nuclear magnetic resonance and X‐ray protein structures of the N‐terminal substrate recognition domain of FimD (FimDN) before and after binding of a chaperone–subunit complex. FimDN consists of a flexible N‐terminal segment of 24 residues, a structured core with a novel fold, and a C‐terminal hinge segment. In the ternary complex, residues 1–24 of FimDN specifically interact with both FimC and the subunit, acting as a sensor for loaded FimC molecules. Together with in vivo complementation studies, we show how this mechanism enables recognition and discrimination of different chaperone–subunit complexes by bacterial pilus assembly platforms.

Automated sequence-specific protein NMR assignment using the memetic algorithm MATCH

MATCH (Memetic Algorithm and Combinatorial Optimization Heuristics) is a new memetic algorithm for automated sequence-specific polypeptide backbone NMR assignment of proteins. MATCH employs local optimization for tracing partial sequence-specific assignments within a global, population-based search environment, where the simultaneous application of local and global optimization heuristics guarantees high efficiency and robustness. MATCH thus makes combined use of the two predominant concepts in use for automated NMR assignment of proteins. Dynamic transition and inherent mutation are new techniques that enable automatic adaptation to variable quality of the experimental input data. The concept of dynamic transition is incorporated in all major building blocks of the algorithm, where it enables switching between local and global optimization heuristics at any time during the assignment process. Inherent mutation restricts the intrinsically required randomness of the evolutionary algorithm to those regions of the conformation space that are compatible with the experimental input data. Using intact and artificially deteriorated APSY-NMR input data of proteins, MATCH performed sequence-specific resonance assignment with high efficiency and robustness.