Pseudocontact Shift-Driven Iterative Resampling for 3D Structure Determinations of Large Proteins

Pseudocontact shifts (PCSs) induced by paramagnetic lanthanides produce pronounced effects in nuclear magnetic resonance spectra, which are easily measured and which deliver valuable long-range structure restraints. Even sparse PCS data greatly enhance the success rate of 3D (3-dimensional) structure predictions of proteins by the modeling program Rosetta. The present work extends this approach to 3D structures of larger proteins, comprising more than 200 residues, which are difficult to model by Rosetta without additional experimental restraints. The new algorithm improves the fragment assembly method of Rosetta by utilizing PCSs generated from paramagnetic lanthanide ions attached at four different sites as the only experimental restraints. The sparse PCS data are utilized at multiple stages, to identify native-like local structures, to rank the best structural models and to rebuild the fragment libraries. The fragment libraries are refined iteratively until convergence. The PCS-driven iterative resampling algorithm is strictly data dependent and shown to generate accurate models for a benchmark set of eight different proteins, ranging from 100 to 220 residues, using solely PCSs of backbone amide protons.Financial support to T.H. and G.O. by the Australian Research Council is gratefully acknowledged

Capturing conformational states in proteins using sparse paramagnetic NMR data

Capturing conformational changes in proteins or protein-protein complexes is a challenge for both experimentalists and computational biologists. Solution nuclear magnetic resonance (NMR) is unique in that it permits structural studies of proteins under greatly varying conditions, and thus allows us to monitor induced structural changes. Paramagnetic effects are increasingly used to study protein structures as they give ready access to rich structural information of orientation and long-range distance restraints from the NMR signals of backbone amides, and reliable methods have become available to tag proteins with paramagnetic metal ions site-specifically and at multiple sites. In this study, we show how sparse pseudocontact shift (PCS) data can be used to computationally model conformational states in a protein system, by first identifying core structural elements that are not affected by the environmental change, and then computationally completing the remaining structure based on experimental restraints from PCS. The approach is demonstrated on a 27 kDa two-domain NS2B-NS3 protease system of the dengue virus serotype 2, for which distinct closed and open conformational states have been observed in crystal structures. By changing the input PCS data, the observed conformational states in the dengue virus protease are reproduced without modifying the computational procedure. This data driven Rosetta protocol enables identification of conformational states of a protein system, which are otherwise difficult to obtain either experimentally or computationally.This study was supported by the Australian Research Council (DP120100561, DP150100383), which the authors gratefully acknowledge

Closed state conformation of NS2B determined by GPS-Rosetta.

<p>(A) Scatter plot of 5,000 all-atom structures showing their combined score of weighted PCS + Rosetta energy versus the Cα RMSD of NS2B relative to the crystal structure in the closed conformation (PDB ID 3U1I). The RMSD was calculated for NS2B only (residues 50–87 of chain A in 3U1I). The conformation selected as the best structure (red point) has the lowest combined energy score and is referred to as the “closed GPS-Rosetta structure”. The four next-lowest combined score structures are represented by blue points. (B) Comparison between GPS-Rosetta structures and the crystal structure. The closed GPS-Rosetta structure is shown in red (NS2B) and grey (NS3pro) and the crystal structure 3U1I is shown in green (NS2B) and grey (NS3pro). The Cα RMSD of NS2B in the closed GPS-Rosetta model is 1.0 Å relative to the crystal structure. The NS2B segments of the next four lowest-energy structures have RMSDs ranging from 0.7 to 2.2 Å and are displayed in different shades of blue. The superimposition shown in the figure used the Cα atoms of NS2B only. (C) Same as (A), except that scoring used PCSs only. (D) Same as (B), except using the structures with the lowest PCS scores identified in (C). In all five models, NS2B has a Cα RMSD between 0.8 and 1.6 Å relative to the NS2B part in the crystal structure.</p

Crystal structures of DENV NS2B-NS3pro and overview of PCSs measured versus the amino acid sequence.

<p>(A) Open state as observed in the ligand-free conformation in the crystal structure 2FOM [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127053#pone.0127053.ref026" target="_blank">26</a>]. NS2B is shown in green and NS3pro is shown in grey. The orange balls identify the locations of residues 34 and 68. We refer to the mutants S34C and S68C as mutants B and C, respectively [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127053#pone.0127053.ref023" target="_blank">23</a>]. To induce PCSs in the protein, the mutants B and C were reacted with a single lanthanide binding tag to form a disulfide bond with either of the cysteine residues at these sites. (B) Closed state as observed in the crystal structure 3U1I [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127053#pone.0127053.ref022" target="_blank">22</a>] with the same color coding as in (A). The closed conformation is presumed to represent the enzymatically active state. (C) Summary of the experimental PCSs. C1 and C2 denote two different lanthanide binding tags used. They differ only in chirality [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127053#pone.0127053.ref023" target="_blank">23</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127053#pone.0127053.ref028" target="_blank">28</a>]. Open circles, filled circles, and boxes identify the residues for which PCSs were observed with Tb³⁺, Tm³⁺, or both Tb³⁺ and Tm³⁺, respectively. The residue numbering used is shown at the top of the amino acid sequence. In this numbering scheme, the mutants B and C are at residues 94 and 129 (highlighted in orange). The green and grey characters identify, respectively, the NS2B and NS3pro segments for which coordinates are reported in the crystal structures (PDB ID: 2FOM, 3U1I). Sequence segments for which no electron density was observed, are highlighted in blue. These parts are probably flexible [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127053#pone.0127053.ref025" target="_blank">25</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127053#pone.0127053.ref027" target="_blank">27</a>].</p

Correlations between experimental and back-calculated PCSs fitted to the closed and open GPS-Rosetta conformations.

<p>To illustrate the information content associated with the PCSs, experimental PCSs measured for the closed state and PCSs generated for the open state (both reported as PCS_exp) were used to fit Δχ-tensors to either the closed or open GPS-Rosetta structures. Subsequently, the Δχ-tensors were used to back-calculate PCS_calc values. Data from Mutant B_C1, Mutant B_C2, Mutant C_C1, and Mutant C_C2 are represented in black, red, cyan, and blue, respectively. Q-factor calculations used the PCSs of backbone amide protons of NS2B only. The correlation plots were produced for all four possible combinations: (A) experimental PCSs for the closed state and closed GPS-Rosetta structure; (B) experimental PCSs of the closed state and open GPS-Rosetta structure; (C) PCSs generated for the open state and closed GPS-Rosetta structure; (D) PCSs generated for the open state and open GPS-Rosetta structure.</p

Density plots illustrating the conformational sampling bias created by PCS data.

<p>(A) Rosetta sampling density using the experimental PCSs of the closed state versus the Cα RMSD of NS2B in the closed state (PDBID 3U1I, chain A, residues 50–87) in black. The corresponding plot versus the Cα RMSD of NS2B in the open state is shown in blue. (B) Same as (A), except that the PCSs used were those calculated for the open state model.</p

Flow chart of calculations.

<p>The flow chart outlines the simulations performed to determine the orientation of NS2B with respect to NS3pro, using different PCS datasets for closed and open conformations.</p

Open state conformation of NS2B generated using GPS-Rosetta.

<p>(A) Scatter plot of 5,000 all-atom structures showing their combined score of weighted PCS + Rosetta energy versus the Cα RMSD of NS2B in the homology model built on the crystal structure 2FOM of the open conformation. The final selected structure (red point) has the lowest combined energy score and is referred to as the “open GPS-Rosetta structure”. The four next-lowest combined score structures are represented by blue points. (B) Superimposition of the best GPS-Rosetta structures onto the homology model. The open GPS-Rosetta structure is shown in red (NS2B) and grey (NS3pro) and the homology model in green (NS2B) and grey (NS3pro). The Cα RMSD of NS2B in the open GPS-Rosetta model is 2.7 Å relative to the homology model. The NS2B segments of the next four lowest-energy structures have RMSDs ranging from 2.7 to 3.1 Å and are displayed in different shades of blue. The superimposition shown in the figure used the Cα atoms of NS2B only. (C) Same as (A), except that scoring used PCSs only. (D) Same as (B), except using the structures with the lowest PCS scores identified in (C). In all five models, NS2B has a Cα RMSD between 2.7 and 3.6 Å relative to the NS2B part in the homology model.</p

Scoring of NS2B conformations of the closed and open state by the Rosetta all-atom energy.

<p>Use of the Rosetta all-atom energy function alone fails to identify the correct structure of the closed state (black points) or the open state (blue points). The lowest-energy structures for both states are shown as red points. Their Cα RMSDs from the corresponding reference structures are about 12 Å. The overall energy landscape is more or less flat in the range from 1 to 15 Å.</p