Sequence-specific NMR Resonance Assignments Research Articles

Sequence-Specific Solution NMR Assignments of the β-Barrel Insertase BamA to Monitor Its Conformational Ensemble at the Atomic Level.

β-barrel outer membrane proteins (Omps) are key functional components of the outer membranes of Gram-negative bacteria, mitochondria, and plastids. In bacteria, their biogenesis requires the β-barrel-assembly machinery (Bam) with the central insertase BamA, but the exact translocation and insertion mechanism remains elusive. The BamA insertase features a loosely closed gating region between the first and last β-strand 16. Here, we describe ∼70% complete sequence-specific NMR resonance assignments of the transmembrane region of the BamA β-barrel in detergent micelles. On the basis of the assignments, NMR spectra show that the BamA barrel populates a conformational ensemble in slow exchange equilibrium, both in detergent micelles and lipid bilayer nanodiscs. Individual conformers can be selected from the ensemble by the introduction of a C-terminal strand extension, single-point mutations, or specific disulfide cross-linkings, and these modifications at the barrel seam are found to be allosterically coupled to sites at the entire barrel circumference. The resonance assignment provides a platform for mechanistic studies of BamA at atomic resolution, as well as for investigating interactions with potential antibiotic drugs and partner proteins.

Complete 1H, 15N and 13C resonance assignments of Bacillus cereus metallo-β-lactamase and its complex with the inhibitor R-thiomandelic acid

β-Lactamases inactivate β-lactam antibiotics by hydrolysis of their endocyclic β-lactam bond and are a major cause of antibiotic resistance in pathogenic bacteria. The zinc dependent metallo-β-lactamase enzymes are of particular concern since they are located on highly transmissible plasmids and have a broad spectrum of activity against almost all β-lactam antibiotics. We present here essentially complete (>96 %) backbone and sidechain sequence-specific NMR resonance assignments for the Bacillus cereus subclass B1 metallo-β-lactamase, BcII, and for its complex with R-thiomandelic acid, a broad spectrum inhibitor of metallo-β-lactamases. These assignments have been used as the basis for determination of the solution structures of the enzyme and its inhibitor complex and can also be used in a rapid screen for other metallo-β-lactamase inhibitors.

X-ray crystallographic and NMR studies of pantothenate synthetase provide insights into the mechanism of homotropic inhibition by pantoate

The structural basis for the homotropic inhibition of pantothenate synthetase by the substrate pantoate was investigated by X-ray crystallography and high-resolution NMR spectroscopic methods. The tertiary structure of the dimeric N-terminal domain of Escherichia coli pantothenate synthetase, determined by X-ray crystallography to a resolution of 1.7 A, showed a second molecule of pantoate bound in the ATP-binding pocket. Pantoate binding to the ATP-binding site induced large changes in structure, mainly for backbone and side chain atoms of residues in the ATP binding HXGH(34-37) motif. Sequence-specific NMR resonance assignments and solution secondary structure of the dimeric N-terminal domain, obtained using samples enriched in (2)H, (13)C, and (15)N, indicated that the secondary structural elements were conserved in solution. Nitrogen-15 edited two-dimensional solution NMR chemical shift mapping experiments revealed that pantoate, at 10 mm, bound at these two independent sites. The solution NMR studies unambiguously demonstrated that ATP stoichiometrically displaced pantoate from the ATP-binding site. All NMR and X-ray studies were conducted at substrate concentrations used for enzymatic characterization of pantothenate synthetase from different sources [Jonczyk R & Genschel U (2006) J Biol Chem 281, 37435-37446]. As pantoate binding to its canonical site is structurally conserved, these results demonstrate that the observed homotropic effects of pantoate on pantothenate biosynthesis are caused by competitive binding of this substrate to the ATP-binding site. The results presented here have implications for the design and development of potential antibacterial and herbicidal agents.

Sequence-specific NMR resonance assignments for human interleukin-5.

Sequence-specific NMR resonance assignments for human interleukin-5.

Structure and dynamics of a denatured 131-residue fragment of staphylococcal nuclease: a heteronuclear NMR study.

A partially folded form of staphylococcal nuclease has been obtained by deleting residues 4-12 and 141-149 of the 149-residue wild-type protein. Sequence-specific NMR resonance assignments have been obtained for 106 of the 131 residues in this protein fragment by using multi-dimensional triple resonance NMR of samples enriched with 13C and 15N. Residues corresponding to helix 2 (residues 98-106) and helix 1 (residues 54-68) of the native state give chemical shifts and NOE effects characteristic of helical structure. These same residues, however, give coupling constants and NOE effects indicative of fast conformational averaging between helical and extended conformations. The residual helix structure observed in the nuclease fragment is thus considerably less persistent than the corresponding structure in the native state. Based on H alpha chemical shifts, we estimate the fractional population of helical conformers to be 30% for helix 2 and 10% for helix 1. Two segments, 83-86 and 94-97, show NOE effects, coupling constants, and lowered amide temperature coefficients consistent with a native-like reverse-turn structure. The C-terminal alpha-helix as well as the fourth and fifth strands of the 5-strand beta-barrel show little evidence for ordered structure. The first three strands of the beta-sheet, part of the catalytic loop, and the first turn of helix 3 give significantly poorer NMR data than the rest of the protein, possibly as a result of exchange broadening, and could not be characterized in detail. That the most persistent elements of structure in the fragment are native-like suggests that nuclease may fold by a hierarchical mechanism.