13 research outputs found
Solution NMR Experiment for Measurement of <sup>15</sup>N–<sup>1</sup>H Residual Dipolar Couplings in Large Proteins and Supramolecular Complexes
NMR
residual dipolar couplings (RDCs) are exquisite probes of protein
structure and dynamics. A new solution NMR experiment named 2D SE2 <i>J</i>-TROSY is presented to measure N–H RDCs for proteins
and supramolecular complexes in excess of 200 kDa. This enables validation
and refinement of their X-ray crystal and solution NMR structures
and the characterization of structural and dynamic changes occurring
upon complex formation. Accurate N–H RDCs were measured at
750 MHz <sup>1</sup>H resonance frequency for 11-mer 93 kDa <sup>2</sup>H,<sup>15</sup>N-labeled Trp RNA-binding attenuator protein tumbling
with a correlation time Ï„<sub>c</sub> of 120 ns. This is about
twice as long as that for the most slowly tumbling system, for which
N–H RDCs could be measured, so far, and corresponds to molecular
weights of ∼200 kDa at 25 °C. Furthermore, due to the
robustness of SE2 <i>J</i>-TROSY with respect to residual <sup>1</sup>H density from exchangeable protons, increased sensitivity
at <sup>1</sup>H resonance frequencies around 1 GHz promises to enable
N–H RDC measurement for even larger systems
Highly Precise Measurement of Kinetic Isotope Effects Using <sup>1</sup>H‑Detected 2D [<sup>13</sup>C,<sup>1</sup>H]-HSQC NMR Spectroscopy
A new method is presented for measuring kinetic isotope
effects
(KIEs) by <sup>1</sup>H-detected 2D [<sup>13</sup>C,<sup>1</sup>H]-heteronuclear
single quantum coherence (HSQC) NMR spectroscopy. The high accuracy
of this approach was exemplified for the reaction catalyzed by glucose-6-phosphate
dehydrogenase by comparing the 1-<sup>13</sup>C KIE with the published
value obtained using isotope ratio mass spectrometry. High precision
was demonstrated for the reaction catalyzed by 1-deoxy-d-xylulose-5-phosphate
reductoisomerase from Mycobacterium tuberculosis. 2-, 3-, and 4-<sup>13</sup>C KIEs were found to be 1.0031(4), 1.0303(12),
and 1.0148(2), respectively. These KIEs provide evidence for a cleanly
rate-limiting retroaldol step during isomerization. The high intrinsic
sensitivity and signal dispersion of 2D [<sup>13</sup>C,<sup>1</sup>H]-HSQC offer new avenues to study challenging systems where low
substrate concentration and/or signal overlap impedes 1D <sup>13</sup>C NMR data acquisition. Moreover, this approach can take advantage
of highest-field spectrometers, which are commonly equipped for <sup>1</sup>H detection with cryogenic probes
Glycosylation Promotes the Random Coil to Helix Transition in a Region of a Protist Skp1 Associated with F‑Box Binding
Cullin-ring-ligases
mediate protein polyubiquitination, a signal
for degradation in the 26S proteasome. The CRL1 class consists of
Skp1/cullin-1/F-box protein/Rbx1 (SCF) complexes that cyclically associate
with ubiquitin-E2 to build the polyubiquitin chain. Within the SCF
complex, the 162-amino acid DdSkp1 from <i>Dictyostelium</i> bridges cullin-1 with an F-box protein (FBP), the specificity factor
for substrate selection. The hydroxylation-dependent glycosylation
of Pro143 of DdSkp1 by a pentasaccharide forms the basis of a novel
O<sub>2</sub>-sensing mechanism in the social amoeba <i>Dictyostelium</i> and other protists. Previous evidence indicated that glycosylation
promotes increased α-helical content correlating with enhanced
interaction with three F-box proteins. To localize these differences,
we used nuclear magnetic resonance (NMR) methods to compare nonglycosylated
DdSkp1 and a glycoform with a single GlcNAc sugar (Gn-DdSkp1). We
report NMR assignments of backbone <sup>1</sup>H<sup>N</sup>, <sup>15</sup>N, <sup>13</sup>C<sup>α</sup>, and <sup>13</sup>CO
nuclei as well as side-chain <sup>13</sup>C<sup>β</sup> and
methyl <sup>13</sup>C/<sup>1</sup>H nuclei of IleÂ(δ1), Leu,
and Val in both unmodified DdSkp1 and Gn-DdSkp1. The random coil index
and <sup>15</sup>NÂ{<sup>1</sup>H} HNOE indicate that the C-terminal
region, which forms a helix–loop–helix motif centered
on Pro143 at the crystallographically defined binding interface with
F-box domains, remains dynamic in both DdSkp1 and Gn-DdSkp1. Chemical
shifts indicate that the variation of conformation in Gn-DdSkp1, relative
to DdSkp1, is limited to this region and characterized by increased
helical fold. Extension of the glycan chain results in further changes,
also limited to this region. Thus, glycosylation may control F-box
protein interactions via a local effect on DdSkp1 conformation, by
a mechanism that may be general to many unicellular eukaryotes
Solution NMR Structure of Yeast Succinate Dehydrogenase Flavinylation Factor Sdh5 Reveals a Putative Sdh1 Binding Site
The yeast mitochondrial protein Sdh5 is required for
the covalent
attachment of flavin adenine dinucleotide (FAD) to protein Sdh1, a
subunit of the heterotetrameric enzyme succinate dehydrogenase. The
NMR structure of Sdh5 represents the first eukaryotic structure of
Pfam family PF03937 and reveals a conserved surface region, which
likely represents a putative Sdh1–Sdh5 interaction interface.
Point mutations in this region result in the loss of covalent flavinylation
of Sdh1. Moreover, chemical shift perturbation measurements showed
that Sdh5 does not bind FAD <i>in vitro</i>, indicating
that it is not a simple cofactor transporter <i>in vivo</i>
Structural and Functional Characterization of DUF1471 Domains of <i>Salmonella</i> Proteins SrfN, YdgH/SssB, and YahO
<div><p>Bacterial species in the Enterobacteriaceae typically contain multiple paralogues of a small domain of unknown function (DUF1471) from a family of conserved proteins also known as YhcN or BhsA/McbA. Proteins containing DUF1471 may have a single or three copies of this domain. Representatives of this family have been demonstrated to play roles in several cellular processes including stress response, biofilm formation, and pathogenesis. We have conducted NMR and X-ray crystallographic studies of four DUF1471 domains from <i>Salmonella</i> representing three different paralogous DUF1471 subfamilies: SrfN, YahO, and SssB/YdgH (two of its three DUF1471 domains: the N-terminal domain I (residues 21–91), and the C-terminal domain III (residues 244–314)). Notably, SrfN has been shown to have a role in intracellular infection by <i>Salmonella</i> Typhimurium. These domains share less than 35% pairwise sequence identity. Structures of all four domains show a mixed α+β fold that is most similar to that of bacterial lipoprotein RcsF. However, all four DUF1471 sequences lack the redox sensitive cysteine residues essential for RcsF activity in a phospho-relay pathway, suggesting that DUF1471 domains perform a different function(s). SrfN forms a dimer in contrast to YahO and SssB domains I and III, which are monomers in solution. A putative binding site for oxyanions such as phosphate and sulfate was identified in SrfN, and an interaction between the SrfN dimer and sulfated polysaccharides was demonstrated, suggesting a direct role for this DUF1471 domain at the host-pathogen interface.</p></div
Addition of ligands to SrfN.
<p><b>A</b>: SrfN–sucrose octasulfate titration monitored with 2-D <sup>1</sup>H-<sup>15</sup>N HSQC. Superimposed spectra: blue, SrfN only; green and red, SrfN +5x and 10x molar excess sucrose octasulfate. <b>B</b>: Chemical shift perturbations following sucrose octasulfate (shown at right) addition mapped onto SrfN surface; the perspective is the same as in Fig. 2 where the positively-charged surface is shown (blue). Ribbon cartoon of SrfN from the same perspective is shown adjacent to the surface depiction. Side chains colored violet have >1 linewidth shift with sucrose octasulfate and similar shifts with heparin & high [SO<sub>4</sub><sup>2−</sup>] but are not conserved in SssB-III (Q24, Q28, A76). Side chains colored magenta have >1 linewidth shift and are conserved in SssB-III (K27/253, H73/294, E89/D311). A fourth conserved residue at the SO<sub>4</sub>-binding position (Y91/313) from the SssB crystal structure does not show chemical shift perturbation upon sucrose octasulfate addition to SrfN. The sulfate ion was positioned by a superposition of the SssB-III crystal structure on SrfN. <b>C</b>: Chemical shift perturbations in SssB-III upon titration of sucrose octasulfate, showing that interactions occur not at the sulfate-binding site common to SrfN, but at a patch of basic residues some distance away.</p
X-ray data collection and refinement statistics for SssB-III.
a<p>Values in parentheses correspond to the highest resolution shell.</p>b<p>R<sub>merge</sub> = Σ<sub>h</sub>Σ<sub>j</sub>|I<sub>hj</sub>–h>|/Σ<sub>h</sub>Σ<sub>j</sub>I<sub>hj</sub>, where I<sub>hj</sub> is the intensity of observation j of reflection h.</p>c<p>R = Σ<sub>h</sub>|F<sub>o</sub>|–|F<sub>c</sub>|/Σ<sub>h</sub>|F<sub>o</sub>| for all reflections, where F<sub>o</sub> and F<sub>c</sub> are observed and calculated structure factors, respectively. R<sub>free</sub> is calculated analogously for the test reflections, randomly selected and excluded from the refinement.</p
Ligand binding sites predicted by coarse-grained simulations.
<p>Predicted sucrose octasulfate interactions with (<b>A</b>) SrfN and with (<b>B</b>) SssB-III, and (<b>C</b>) predicted maltohexaose dodecasulfate interactions with SrfN. Red, yellow, and green indicate binding score levels of >0.1, >0.05, and >0.02, respectively. <b>D</b> and <b>E</b>: Low energy structures of models shown in panels A and B after being reverse-mapped to atomistic sucrose octasulfate in the space occupied by the coarse-grained equivalent, followed by 1000 steps of vacuum minimization in GROMACS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101787#pone.0101787-Hess1" target="_blank">[73]</a> using the CHARMM force field to eliminate clashes, with ligand parameters were derived using SwissParam <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101787#pone.0101787-Zoete1" target="_blank">[74]</a>. The all-atom models are shown for illustrative purposes and do not necessarily indicate global free energy minima at all-atom resolution.</p
Protein structures from the Protein Data Bank that are similar (Z score >5.0) to DUF1471 proteins, determined with Dali [20]. Structures are X-ray structures except as noted.
<p>Protein structures from the Protein Data Bank that are similar (Z score >5.0) to DUF1471 proteins, determined with Dali <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101787#pone.0101787-Holm1" target="_blank">[20]</a>. Structures are X-ray structures except as noted.</p
DUF 1471 sequences.
<p><b>A</b>: Multiple sequence alignment of DUF1471 paralogues from <i>S.</i> Typhimurium, as well as <i>E. coli</i> YbiM, for which there is no close homolog in <i>Salmonella</i>. Alignment of SrfN, YahO, SssB-I and SssB-III is structure-based over the entire structured sequence (SrfN residues 22–91), other alignments are sequence-based and are between core regions only (SrfN residues 35–91) because sequence identity to SrfN residues 22–38 is low and alignments in this region are uncertain. Secondary structure in SrfN, YahO, SssB-I, and SssB-III is indicated above: E = extended (β-sheet) structure, H = helix. The core residues of the sulfate-binding motif in SrfN are indicated with asterisks. Conserved sequence motifs identified by Rudd <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101787#pone.0101787-Rudd1" target="_blank">[1]</a> are underlined. Other conserved residues are highlighted in green or dark grey. Two notable loop regions in the structure are also indicated. SrfN and YahO both have C-terminal tag sequences LEHHHHHH that are not shown. Light grey highlighted portions indicate likely signal sequences for periplasmic localization that are known or likely to be cleaved by a signal peptidase. In the case of SrfN and YahO, the signal sequence was proven experimentally to be cleaved during heterologous expression in <i>E. coli</i>. Inter-domain regions of SssB are not shown. Lower case letters in SssB-III (C-terminal domain) indicate residues with missing electron density in the X-ray structure. Highly conserved residues are indicated by highlighting (blue = hydrophobic, green = polar), somewhat conserved residues are indicated with grey highlighting. The following sequences are listed (<i>S.</i> Typhimurium LT2 locus and UniProt/TrEMBL numbers in parentheses): SrfN (STM0082/Q7CR88), YjfY (STM4389/Q8ZK84), YhcN (STM3361/Q8ZLP6), YcfR seq. I (STM1214/Q8ZQ03), YcfR seq. II (STM3362/Q7CPN0), YahO (STM0366/Q7CR49), YbiJ (STM0823/Q7CQW3), YkgI (STM0565/Q7CR04), YjfO (STM4379/Q8ZK92), YjfN (STM4378/Q8ZK93), SssB (STM1478/Q8ZPL1), YbiM/McbA (<i>E. coli</i>, P0AAX6). <b>B</b>: Unrooted phylogenetic tree (phylogram) constructed from ten diverse genera from the Enterobacteriaceae. Major branches containing <i>Salmonella</i> and <i>E. coli</i> subfamily members are indicated. <b>C</b>: Multiple sequence alignment of SrfN homologues: a subfamily of DUF1471 proteins. For each sequence and abbreviated organism name listed, the full genus and species name, protein/ORF name, database accession number, and similarity to SrfN, excluding the signal sequence, are as follows: Sty, <i>Salmonella enterica</i> Typhimurium, STM0082 (SrfN), NP_459087 and many other <i>Salmonella</i> strains; Sbo, <i>Salmonella bongori</i>, SBG_0068, YP_004728986 (93%); Cro, <i>Citrobacter rodentium</i>, ROD_12311, YP_003364817 (80%); Eho, <i>Enterobacter hormaechei</i>, HMPREF9086_0329, ZP_08496071 (65%); Eae, <i>Enterobacter aerogenes</i> EAE_13230, YP_004592839 (70%); Kpn, <i>Klebsiella pneumonia</i>, KPK_4095, YP_002239898 (68%); Pan, <i>Pantoea</i> sp., Pat9b_3745, YP_004117591 (61%). Notes: Other <i>Salmonella, Klebsiella,</i> and <i>Enterobacter</i> species and strains contain identical or nearly identical sequences to the representatives shown here. However, some <i>Pantoea</i> species do not contain homologues that fall within this DUF1471 subfamily.</p