Three-dimensional structure of the weakly associated protein homodimer SeR13 using RDCs and paramagnetic surface mapping

Abstract

The traditional NMR-based method for determining oligomeric protein structure usually involves distinguishing and assigning intra- and intersubunit NOEs. This task becomes challenging when determining symmetric homo-dimer structures because NOE cross-peaks from a given pair of protons occur at the same position whether intra- or intersubunit in origin. While there are isotope-filtering strategies for distinguishing intra from intermolecular NOE interactions in these cases, they are laborious and often prove ineffectual in cases of weak dimers, where observation of intermolecular NOEs is rare. Here, we present an efficient procedure for weak dimer structure determination based on residual dipolar couplings (RDCs), chemical shift changes upon dilution, and paramagnetic surface perturbations. This procedure is applied to the Northeast Structural Genomics Consortium protein target, SeR13, a negatively charged Staphylococcus epidermidis dimeric protein (K(d) 3.4 ± 1.4 mM) composed of 86 amino acids. A structure determination for the monomeric form using traditional NMR methods is presented, followed by a dimer structure determination using docking under orientation constraints from RDCs data, and scoring under residue pair potentials and shape-based predictions of RDCs. Validation using paramagnetic surface perturbation and chemical shift perturbation data acquired on sample dilution is also presented. The general utility of the dimer structure determination procedure and the possible relevance of SeR13 dimer formation are discussed.

Figure 1

The ensemble of 10 structures with the least distance violations showing the monomeric form of SeR13 calculated using XPLOR-NIH. The coordinates for SeR13 have been deposited in the protein data bank with an accession code of 2K1H. The colors in the display progress from blue to red based on sequence.

Figure 2

Concentration dependent chemical shift studies of SeR13. (A) The stacked ¹⁵N-¹H HSQC spectra of SeR13 with protein concentration of 0.025, 0.5, 1.0, 1.5, and 2.1 mM are shown in red, orange, yellow, magenta, and cyan, respectively. The self-dissociation constant of SeR13 was obtained by monitoring the chemical shift changes as a function of protein concentrations. The fitting of chemical shifts assumes a 1:1 protein-protein complex. (B) ¹H of L19 and (C) ¹⁵N of Y56.

Figure 3

(A) The combined ¹⁵N and ¹H chemical shifts perturbations of each resonance for SeR13 between 0.1 and 2.1 mM. (B) The backbone of residues with amide chemical shift perturbations > 0.1 Δ_ppm are shown in red (L19, N26, S27, F28, T29, Y56, D59, F60, I61, S62, and I63). The backbone of residues with amide chemical shift perturbations of < 0.1 but greater than 0.04 Δ_ppm are shown in orange (I4, T9, K16, S18, T29 and D59). Residues that show chemical shifts changes but are overlapped with another crosspeaks are shown in black.

Figure 4

(A) Comparison plot of protection from paramagnetic relaxation enhancement on increasing SeR13 concentration from 0.1 to 1.9 mM in the presence of 1.0 and 2.5 mM Gd-DTPA, respectively. (B) Regions where the protection factor decreased by more than one standard deviation are shown in blue (A33, A34, G37, E49, G50, K52, A69, W71, and N72) and regions where protection increased by more than 1 standard deviation are shown in red (V17, L19, S20, Y56, V57, D59, I61, and I63).

Figure 5

RDCs for the dimer of SeR13 by data extrapolation. (A) The RDCs values were extrapolated using data points collected with different protein concentrations. (B) The RDCs correlation plot between the measured or projected dimer RDCs against the computed RDCs using the monomer NMR structure.

Figure 6

Alignment axis directions for SeR13. (A) The possible principal order tensor solutions of SeR13 for bicelle and Gel are plotted onto the Sauson-Flamsteed projection grid. (B) The directions of the order tensors are plotted onto the molecular frame of SeR13 to illustrate the symmetry axis between the two alignment media.

Figure 7

The dimeric models of SeR13. (A) The ensemble of 10 structures with lowest energies. (B) The hydrophobic interactions between the sidechains of F28, V57, and F60 of subunit A (red) and subunit B (blue) are shown in ball and stick representations. (C) The ionic interactions between sidechains of K16 and R23 of one subunit to the sidechain of D59 and D64 of the other subunit are shown in ball and stick representations.