Solution NMR Experiment for Measurement of ¹⁵N–¹H 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 ¹H resonance frequency for 11-mer 93 kDa ²H,¹⁵N-labeled Trp RNA-binding attenuator protein tumbling with a correlation time τ_c 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 ¹H density from exchangeable protons, increased sensitivity at ¹H resonance frequencies around 1 GHz promises to enable N–H RDC measurement for even larger systems

Highly Precise Measurement of Kinetic Isotope Effects Using ¹H‑Detected 2D [¹³C,¹H]-HSQC NMR Spectroscopy

A new method is presented for measuring kinetic isotope effects (KIEs) by ¹H-detected 2D [¹³C,¹H]-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 1-¹³C 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 4-¹³C 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 [¹³C,¹H]-HSQC offer new avenues to study challenging systems where low substrate concentration and/or signal overlap impedes 1D ¹³C NMR data acquisition. Moreover, this approach can take advantage of highest-field spectrometers, which are commonly equipped for ¹H detection with cryogenic probes

Statistics of hMcl-1(171–327) NMR Structure.

a<p>Related to pairs with non-degenerate chemical shift.</p>b<p>Regular secondary element: α-helical residues 173–191, 204–235, 240–253, 262–280, 284–301, 303–308 and 311–319.</p>c<p>Ordered residues: 172–192,194–198, 204–235, 238–255, 262–321 with dihedral angle order parameters S(φ) and S(ψ) > 0.90. Z-scores were computed relative to corresponding structure quality measures for high resolution X-ray crystal structures <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Bhattacharya1" target="_blank">[42]</a>.</p

Superposition of selected Mcl-1 structures.

<p>(A) Structures of hMcl-1(171–327) (green) and mMcl-1(152–308) (cyan, PDB accession code 1wsx) after superposition of the backbone N, C^α and C’ atoms of the α-helices for minimal rmsd. (B) Ribbon drawing (zoomed into (A)) showing the different binding groove widths of human (green) and mouse (cyan) protein. The distances between the Cα-atoms of residues His 224 in helix α2 (His 205 in mMcl-1) and His 252 (His 233 in mMcl-1) at the C-terminus of helix α3 are highlighted: ∼16 Å in hMcl-1(171–327) and ∼14 Å mMcl-1(152–308) (C) Superposition as in (A) of hMcl-1(171–327) (green) and mMcl-1(152–308) (cyan, 1wsx), and six selected Mcl-1 complex structures (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone-0096521-t002" target="_blank">Table 2</a>): human Mcl-1 complexed with Bim BH3 (magenta, 2nla); chimeric rat-human rMcl-1(171–208)hMcl-1(209–327) complexed with mouse mNoxaB BH3 (yellow, 2rod); mouse mMcl-1(152–308) complexed with mouse NoxaA BH3 (pink, 2roc); mouse mMcl-1(152–308) complexed with mouse Puma BH3 (grey, 2jm6); mouse mMcl-1(152–308) complexed with mouse NoxaB BH3 (purple, 2rod); chimeric rat-human mMcl-1(171–208)hMcl-1(209–327) complexed with human Bim BH3 (orange, 2nl9); chimeric rat-human mMcl-1(171–208)hMcl-1(209–327) complexed with human Bim BH3(L62A, F68A) (light green, 3d7v).The figures were prepared with the programs MOLMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Koradi1" target="_blank">[36]</a> and PYMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Delano1" target="_blank">[37]</a>.</p

Rmsd values for comparison of the NMR structure of hMcl-1(171–327) with the structures of mouse mMcl-1(152–308) and Mcl-1complexes.^a

a<p>Average pairwise rmsd values (Å) were calculated for backbone heavy atoms N, C^α, and C’ between the 20 conformers of Mcl-1(171–327) and corresponding polypeptide segments in the other structures. The distances dCA (in Å) between the C^α-atoms of residues His 224 in helix α2 (His 205 in mMcl-1) and His 252 (His 233 in mMcl-1) at the C-terminus of helix α3 are provided as a measure for the width of the BH3 binding groove.</p>b<p>Residue numbers are for hMcl-1(171–327); residues 194–202 were excluded since one structure (2nl9^k) does not contain the corresponding residues; residues 172–193 and 203–321 correspond to residues 153–174 and 184–302 in mMcl-1, and residues 209–321 correspond to residues 190–302 in mMcl-1.</p>c<p>Helices α1–α7 in hMcl-1 comprise residues 173–191, 204–235, 240–253, 262–280, 284–301, 303–308 and 311–319; the corresponding residues in mMcl-1 are: 155–172, 185–216, 221–234, 243–261, 265–282, 284–289 and 292–300.</p>d<p>Helices α2–α7 in hMcl-1 and residues 204–208 (numbers in hMcl-1) were excluded</p>e<p>Mouse mMcl-1(152–308), PDB accession code 1wsx (the mean NMR coordinates were used) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Day1" target="_blank">[10]</a>.</p>f<p>Human hMcl-1 complexed with human hBim BH3, 2pqk <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Fire1" target="_blank">[11]</a>.</p>g<p>Chimiric rat-human rMcl-1(171–208)hMcl-1(209–327) complexed with mouse mNoxaB BH3, 2nla <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Czabotar1" target="_blank">[9]</a>.</p>h<p>Mouse mMcl-1 complexed with mouse mNoxaA BH3, 2rod <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Day2" target="_blank">[12]</a>.</p>i<p>Mouse mMcl-1 complexed with mouse mPuma BH3, 2roc <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Day2" target="_blank">[12]</a>.</p>j<p>Mouse mMcl-1 complexed with mouse mNoxaB BH3, 2jm6 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Czabotar1" target="_blank">[9]</a>.</p>k<p>Chimiric rat-human Mcl-1 complexed with human hBim BH3, 2nl9 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Czabotar1" target="_blank">[9]</a>.</p>l<p>Chimiric rat-human Mcl-1 complexed with human hBim (L62A, F68A), 3d7v <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Lee1" target="_blank">[13]</a>.</p>J<p>Human hMcl1 complexed with human Bid BH3, 2kbw <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Liu1" target="_blank">[15]</a>.</p>k<p>Human hMcl-1 complexed with human Bim BH3 mutant I2dY, 3kj0 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Fire1" target="_blank">[11]</a>.</p>l<p>Human hMcl-1 complexed with human BimL12Y, 3io9 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Lee2" target="_blank">[16]</a>.</p>m<p>Human hMcl1 complexed with human Bim BH3 mutant I2dA, 3kj1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Fire1" target="_blank">[11]</a>.</p>n<p>Human hMcl1 complexed with human Bim BH3 mutant F4aE, 3kj2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Fire1" target="_blank">[11]</a>.</p>o<p>Human hMcl-1 complexed with Mcl1 specific selected peptide B7, 3kz0 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Dutta1" target="_blank">[41]</a>.</p>p<p>Human hMcl-1 complexed with human Mcl1 BH3, 3mk8 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Stewart1" target="_blank">[17]</a>.</p>q<p>Human hMcl1 complexed with human Bax BH3, 3pk1.</p>r<p>Mouse mMcl-1 complexed with mouse Noxa BH3, 4g35 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Muppidi1" target="_blank">[18]</a>.</p>s<p>Human hMcl-1 complexed with 6-chloro-3-[3-(4-chloro-3,5-dimethylphenoxy)propyl]-1H-indole-2-carboxylic acid, 4hw2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Friberg1" target="_blank">[14]</a>.</p>t<p>Human hMcl-1 complexed with 6-chloro-3-[3-(4-chloro-3,5-dimethylphenoxy)propyl]-1H-indole-2-carboxylic acid, 4hw3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Friberg1" target="_blank">[14]</a>.</p>u<p>Human hMcl-1 complexed with human Mcl1 BH3, 4hw4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Friberg1" target="_blank">[14]</a>.</p

NMR structure of hMcl-1(171–327).

<p>(<b>A</b>) Backbone of the 20 CYANA conformers representing the solution structure of hMcl-1(171–327) after superposition of backbone N, C^α and C’ atoms of the α-helices for minimal rmsd. The three BH sequence motifs are colored in green (BH3), red (BH1) and blue (BH2), respectively. (<b>B</b>) Ribbon drawing of the lowest energy conformer of hMcl-1(171–327). α-helices α1-α7 are labeled and colored differently, and the N- and C-termini are labeled as “<i>N’</i> ” and “<i>C’</i> ”. The figures were generated using the programs MOLMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Koradi1" target="_blank">[36]</a> and PYMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Delano1" target="_blank">[37]</a>.</p

Electrostatic surface potentials.

<p>(<b>A</b>) For human hMcl-1(171–327) in the orientation shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone-0096521-g001" target="_blank">Figure 1</a> (left) and after rotation by 180° about the vertical axis (right). Surface colors (blue for positively charged; red for negatively charged) indicated the electrostatic potential calculated by using PYMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Delano1" target="_blank">[37]</a> and its default vacuum electrostatics protocol. (<b>B</b>) Same as in (A) but for mouse mMcl-1(152–308).</p

Metal-Mediated Affinity and Orientation Specificity in a Computationally Designed Protein Homodimer

Computationally designing protein–protein interactions with high affinity and desired orientation is a challenging task. Incorporating metal-binding sites at the target interface may be one approach for increasing affinity and specifying the binding mode, thereby improving robustness of designed interactions for use as tools in basic research as well as in applications from biotechnology to medicine. Here we describe a Rosetta-based approach for the rational design of a protein monomer to form a zinc-mediated, symmetric homodimer. Our metal interface design, named MID1 (NESG target ID OR37), forms a tight dimer in the presence of zinc (MID1-zinc) with a dissociation constant <30 nM. Without zinc the dissociation constant is 4 μM. The crystal structure of MID1-zinc shows good overall agreement with the computational model, but only three out of four designed histidines coordinate zinc. However, a histidine-to-glutamate point mutation resulted in four-coordination of zinc, and the resulting metal binding site and dimer orientation closely matches the computational model (Cα rmsd = 1.4 Å)

Enzyme Engineering Based on X‑ray Structures and Kinetic Profiling of Substrate Libraries: Alcohol Dehydrogenases for Stereospecific Synthesis of a Broad Range of Chiral Alcohols

The narrow substrate scope of naturally occurring alcohol dehydrogenases (ADHs) greatly limits the enzymatic synthesis of important chiral alcohols. On the basis of X-ray crystal structures and kinetic profiling of a substrate library, we engineered variants of the stereospecific alcohol dehydrogenase from Candida parapsilopsis. This resulted in a set of four mutant enzymes which enable the asymmetric reduction of a broad range of prochiral ketones, including valuable pharmaceuticals and fine chemicals. The engineering strategy of this study paves the way for creating additional ADHs tailored for production of complex chiral alcohols

Schematic representation of secondary structure element topologies.

<p>(A) YxeF, (B) lipocalins and (C) fatty acid-binding proteins. β-strands are represented by arrows, α-helices by rectangles, and 3₁₀-helices by ellipses. N- and C-termini are indicated as N and C respectively, and the ‘Ω-type’ loop L1 shared by YxeF and lipocalins is labeled.</p