Chenyun Guo

Structural basis for targeting the ribosomal protein S1 of Mycobacterium tuberculosis by pyrazinamide.

Pyrazinamide (PZA) is a first‐line drug for tuberculosis (TB) treatment and is responsible for shortening the duration of TB therapy. The mode of action of PZA remains elusive. RpsA, the ribosomal protein S1 of Mycobacterium tuberculosis (Mtb), was recently identified as a target of PZA based on its binding activity to pyrazinoic acid (POA), the active form of PZA. POA binding to RpsA led to the inhibition of trans‐translation. However, the nature of the RpsA–POA interaction remains unknown. Key questions include why POA exhibits an exquisite specificity to RpsA of Mtb and how RpsA mutations confer PZA resistance. Here, we report the crystal structures of the C‐terminal domain of RpsA of Mtb and its complex with POA, as well as the corresponding domains of two RpsA variants that are associated with PZA resistance. Structural analysis reveals that POA binds to RpsA through hydrogen bonds and hydrophobic interactions, mediated mainly by residues (Lys303, Phe307, Phe310 and Arg357) that are essential for tmRNA binding. Conformational changes induced by mutation or sequence variation at the C‐terminus of RpsA abolish the POA binding activity. Our findings provide insights into the mode of action of PZA and molecular basis of PZA resistance associated with RpsA mutations.

Methyl-detected ‘out-and-back’ NMR experiments for simultaneous assignments of Alaβ and Ileγ2 methyl groups in large proteins

A set of sensitive methyl-detected ‘out-and-back’ NMR experiments for simultaneous assignments of Alaβ and Ileγ2 methyl positions in large proteins is described. The developed methodology is applied to an 82-kDa enzyme Malate Synthase G. Complete alanine β and isoleucine γ2 1H–13C methyl chemical shift assignments could be obtained from the set of new methyl-detected ‘out-and-back’ 3D experiments. The described methodology for methyl assignments should be applicable to protein molecules within approximately 100-kDa molecular weight range irrespective of the labeling strategy chosen to produce selectively protonated Alaβ and Ileγ2 13CH3 sites on a deuterated background.

An NMR Experiment for Simultaneous TROSY-Based Detection of Amide and Methyl Groups in Large Proteins

A sensitive 2D NMR experiment for simultaneous time-shared TROSY-type detection of amide and methyl groups in high-molecular-weight proteins is described. The pulse scheme is designed to preserve the slowly decaying components of both 1H-15N and methyl 13CH3 spin systems in the course of indirect evolution and acquisition periods. The proposed methodology is applied to the study of substrate binding to {U-[15N,2H]; Ile-[13CH3]; Leu,Val-[13CH3/12CD3]}-labeled 82-kDa enzyme Malate Synthase G and is expected to accelerate NMR-based screening of large proteins labeled with 15N and selectively labeled with 13CH3 at methyl sites.

Selective 1H- 13C NMR spectroscopy of methyl groups in residually protonated samples of large proteins.

Methyl 13CHD2 isotopomers of all methyl-containing amino-acids can be observed in residually protonated samples of large proteins obtained from [U-13C,1H]-glucose/D2O-based bacterial media, with sensitivity sufficient for a number of NMR applications. Selective detection of some subsets of methyl groups (Alaβ, Thrγ2) is possible using simple ‘out-and-back’ NMR methodology. Such selective methyl-detected ‘out-and-back’ NMR experiments allow complete assignments of threonine γ2 methyls in residually protonated, [U-13C,1H]-glucose/D2O-derived samples of an 82-kDa enzyme Malate Synthase G. [U-13C,1H]-glucose/D2O-derived protein samples are relatively inexpensive and are usually available at very early stages of any NMR study of high-molecular-weight systems.

Selective backbone labeling of proteins using {1,2-13C2}-pyruvate as carbon source

A simple isotope labeling approach for selective 13C/15N backbone labeling of proteins is described. Using {1,2-13C2}-pyruvate as the sole carbon source in bacterial growth media, selective incorporation of 13Cα-13CO spin-pairs into the backbones of protein molecules with medium-to-high levels of 13C-enrichment is possible for a subset of 12 amino acids. The isotope labeling scheme has been tested on a pair of proteins—a 7-kDa immunoglobulin binding domain B1 of streptococcal protein G and an 82-kDa enzyme malate synthase G. A number of protein NMR applications are expected to benefit from the {1,2-13C2}-pyruvate based protein production.

Identification of HN-methyl NOEs in large proteins using simultaneous amide-methyl TROSY-based detection

A pair of HN-methyl NOESY experiments that are based on simultaneous TROSY-type detection of amide and methyl groups is described. The preservation of cross-peak symmetry in the simultaneous 1H–15N/13CH3 NOE spectra enables straightforward assignments of HN-methyl NOE cross-peaks in large and complex protein structures. The pulse schemes are designed to preserve the slowly decaying components of both 1H–15N and methyl 13CH3 spin-systems in the course of indirect evolution (t2) and acquisition period (t3) of 3D NOESY experiments. The methodology has been tested on {U-[15N,2H]; Ileδ1-[13CH3]; Leu,Val-[13CH3,12CD3]}-labeled 82-kDa enzyme Malate Synthase G (MSG). A straightforward procedure that utilizes the symmetry of NOE cross-peaks in the time-shared 3D NOE data sets allows unambiguous assignments of more than 300 HN-methyl interactions in MSG from a single 3D data set providing important structural restraints for derivation of the backbone global fold.

Effects of removing a conserved disulfide bond on the biological characteristics of rat lipocalin-type prostaglandin D synthase

Three cysteine residues, Cys(65), Cys(89), and Cys(186) in lipocalin-type prostaglandin D synthase (L-PGDS), are conserved among all species and the disulfide bond between Cys(89) and Cys(186) is highly conserved among most, but not all, lipocalins. In this study, four rat L-PGDS variants were constructed by site-directed mutagenesis, and the conserved disulfide bond in several variants was removed by substituting cysteine with alanine. The effects of removing this disulfide bond on their biological characteristics were investigated. The NMR experiments indicated that the removal of disulfide did not change their conformations significantly. However, both thermal-induced and urea-induced unfolding experiments showed that the stabilities of enzymes without the disulfide bond decreased significantly. Moreover, the ligand-binding affinities of these variants were assessed by fluorescence experiments. Dissociation constants (K(d)) of 0.668, 0.689, 0.543 and 0.571 microM were obtained for ANS binding to wild-type rat L-PGDS, C(65)A, C(186)A, and C(89,186)A variants, respectively, and 71.2 and 62.3 nM for retinoic acid binding to wild-type rat L-PGDS and the C(186)A variant, respectively. These results suggested that the removal of the disulfide bond slightly increased the affinities for ligand binding by changing the hydrophobic regions. This study may offer valuable information for further studies on other rat lipocalins.

High resolution measurement of methyl 13C(m)-13C and 1H(m)-13C(m) residual dipolar couplings in large proteins.

NMR methodology is developed for high-resolution, accurate measurements of methyl (1)H(m)-(13)C(m) ((1)D(CH)) and (13)C(m)-(13)C ((1)D(CC)) residual dipolar couplings (RDCs) in ILV-methyl-protonated high-molecular-weight proteins. Both types of RDCs are measured in a three-dimensional (3D) mode that allows dispersion of correlations to the third ((13)C(β/γ)) dimension, alleviating the problem of overlap of methyl resonances in highly complex and methyl-abundant protein structures. The methodology is applied to selectively ILV-protonated 82-kDa monomeric enzyme malate synthase G (MSG) that contains 273 ILV methyl groups with substantial overlap of methyl resonances in 2D methyl (1)H-(13)C correlation maps. A good agreement is observed between the measured RDCs of both types and those calculated from the crystallographic coordinates of MSG for the residues with low-amplitude internal dynamics. Although the measurement of (1)D(CH) RDCs from the acquisition dimension of NMR spectra imposes certain limitations on the accuracy of obtained (1)D(CH) values, (1)D(CH) couplings can be approximately corrected for cross-correlated relaxation effects. The ratios of (1)D(CH) and (1)D(CC) couplings ((1)D(CH)/(1)D(CC)) are independent of methyl axis dynamics and the details of residual alignment [Ottiger, M.; Bax, A. J. Am. Chem. Soc. 1999, 121, 4690.]. The (1)D(CH)/(1)D(CC) ratios obtained in MSG can therefore validate the employed correction scheme.

Recombinant expression and characterization of an epididymis-specific antimicrobial peptide BIN1b/SPAG11E.

BIN1b was reported as an epididymis-specific beta-defensin antimicrobial peptide. In this paper, the recombinant BIN1b was expressed and purified by fusing with GB1-His tag. The size-exclusion gel filtration experiment indicated that the fusion protein GB1-BIN1b formed multimers at pH 7.4, and existed as monomer at pH 4.5. The oligomerization of GB1-BIN1b was only related to pH value, neither to NaCl concentration nor protein concentration. Far-UV circular dichroism (CD) spectra also showed the fusion protein had more ordered secondary structures at pH 4.5 than at pH 7.4, as a negative peak appeared around 218 nm indicative of typical beta-sheet. The 2D (15)N-(1)H heteronuclear single-quantum coherence (HSQC) spectra suggested that the fusion protein adopted a compact three-dimensional structure at pH 4.5. Colony forming unit (CFU) inhibition assay demonstrated that 25 microM fusion protein at pH 7.4 had an antimicrobial activity of 40% against E. coli K(12)D(31), which might imply the fusion protein functions as multimeric states. In conclusion, the GB1 fusion partner helps BIN1b form a stable homogenous conformation to facilitate subsequent structural determination without a significant effect on the antimicrobial activity.

1H, 13C, 15N backbone and side-chain resonance assignments of the human Raf-1 kinase inhibitor protein

Raf-1 kinase inhibitor protein (RKIP) plays a pivotal role in modulating multiple signaling networks. Here we report backbone and side chain resonance assignments of uniformly 15N, 13C labeled human RKIP.