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 6 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.

Glycosylation Alters Dimerization Properties of a Cell-surface Signaling Protein, Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1)

“…They also provided a basis for measurement of rotational correlation times that are sensitive to dimer formation (37), and for the collection of residual dipolar coupling (RDC) data that are orientation dependent. The latter can more rigorously confirm the retention of the domain structure, as well as distinguish which of the dimer structures seen in crystal structures may be present in solution (38,39). Our findings demonstrate that the non-glycosylated form exists as a dimer involving the GFCCЈCЉ interface in solution, but minimal glycosylation inhibits all types of dimerization.…”

Chemokine Oligomerization in Cell Signaling and Migration

“…When sufficient numbers of RDCs are measured for well-defined structures, alignment axes can be determined, and when two different proteins are involved superposition of alignment axes greatly restricts allowed structures. 124,125 Typically, these RDC-based restrictions are combined with data on interfacial contacts and translational restraints to produce unique structures. For symmetric homo-oligomers, there are symmetry-based rules that allow identification of the symmetry axis in the coordinate frame of the monomer and generation of symmetry-related elements by rotation about this axis.…”

The Use of Residual Dipolar Coupling in Studying Proteins by NMR

“…In addition, they can be utilized to establish quaternary structure of symmetric oligomers. Prestegard and coworkers have developed methods to determine dimer structure using RDC data [83, 84]. The underlying principle is that the rotational symmetric C 2 axis of any protein dimer should be parallel to one axis of the alignment tensor of the dimer.…”

“…Prestegard and colleagues further extended this approach to study structures of weakly associated dimer. The Staphylococcus epidermidis proteins SeR13 weakly dimerize at a K d on the order of 10 −3 M [84]. The measured RDCs were the weighted average of monomer and dimer states and the resulting alignment tensor from direct fitting would be the average of both monomer and dimer alignment tensors.…”

“…The measured RDCs were the weighted average of monomer and dimer states and the resulting alignment tensor from direct fitting would be the average of both monomer and dimer alignment tensors. Lee et al [84] circumvented this by deriving the exact K d value from concentration dependent chemical shift values, then extrapolating the RDCs as a function of protein concentration together with the K d to yield pure RDC data set for the dimer. The rest of the work to determine the dimer structure was similar to the previous approach [83], but with the additional restraints from chemical shift perturbation and paramagnetic surface perturbation data that helped identifying newly buried surface residues upon dimerization.…”