Probing large conformational rearrangements in wild-type and mutant spectrin using structural mass spectrometry

Abstract

Conformational changes of macromolecular complexes play key mechanistic roles in many biological processes, but large, highly flexible proteins and protein complexes usually cannot be analyzed by crystallography or NMR. Here, structures and conformational changes of the highly flexible, dynamic red cell spectrin and effects of a common mutation that disrupts red cell membranes were elucidated using chemical cross-linking coupled with mass spectrometry. Interconversion of spectrin between closed dimers, open dimers, and tetramers plays a key role in maintaining red cell shape and membrane integrity, and spectrins in other cell types serve these as well as more diverse functions. Using a minispectrin construct, experimentally verified structures of closed dimers and tetramers were determined by combining distance constraints from zero-length cross-links with molecular models and biophysical data. Subsequent biophysical and structural mass spectrometry characterization of a common hereditary elliptocytosis-related mutation of α-spectrin, L207P, showed that cell membranes were destabilized by a shift of the dimer-tetramer equilibrium toward closed dimers. The structure of αL207P mutant closed dimers provided previously unidentified mechanistic insight into how this mutation, which is located a large distance from the tetramerization site, destabilizes spectrin tetramers and cell membrane integrity.hereditary hemolytic anemia | protein conformations S olving static structures of protein complexes and probing dynamic conformational rearrangements have frequently provided mechanistic insights into macromolecular functions as well as effects of disease-related mutations. However, highresolution structural techniques such as X-ray crystallography and NMR usually cannot be applied to large proteins that are highly flexible, intrinsically disordered, or undergo large conformational changes. Chemical cross-linking coupled with mass spectrometry (CX-MS) is a powerful tool that identifies proximal amino acid residues of proteins in solution. These spatial constraints can greatly enhance and experimentally validate molecular modeling to result in reliable medium-resolution structures. This approach has been effectively used in numerous studies (1-5), although homobifunctional lysine-specific cross-linkers with relatively long spacer arms were mostly used. Such reagents have been preferred because they enable introduction of isotope labels and other functional sites that facilitate cross-linked peptide identifications (6-9). In contrast, zero-length cross-linkers such as 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide form a covalent bond between reactive amines (N-terminal amine or lysine side chain) and carboxyls (C-terminal carboxyl or aspartic or glutamic acid side chains) without inserting extra atoms (Fig. 1). Hence, reactive groups have to be within salt bridge distances to react. This results in tighter distance constraints that outperform those from longer cross-links when these data are used to refine structural model...

Anatomy of the red cell membrane skeleton: unanswered questions

“…The details of spectrin structure are shown in Figure 3. [12][13][14][15][16][17][18][19][20][21][22][23][24][25][26] On average, 6 spectrins bind per actin filament, leading to a pseudohexagonal arrangement. 27 Isolated a-and b-spectrin chains bind to each other electrostatically at a pair of nucleation sites formed by repeats near the spectrin tail ( Figure 3A).…”

“…In spectrin ab dimers, the longer a chain folds back on itself, so the a0/b17 repeat interacts with repeat a4 and the a1 and a3 repeats contact each other ( Figure 3A). 13 In the a 2 b 2 -heterotetramers, there are 3 such side-to-side interactions between the longer a chains ( Figure 3C). These lateral interactions, though weak, contribute to self-association.…”

“…Note that for the spectrin ab dimer to convert to the a 2 b 2 tetramer, it must first cleave the internal linkage between the partial spectrin repeats a0 and b17 and then unfold (open dimer). 13 This is the rate-limiting step in the dimer-tetramer equilibrium.…”

The Physiological Molecular Shape of Spectrin: A Compact Supercoil Resembling a Chinese Finger Trap

“…This model is morphologically similar to the rotary shadowed electron micrographs of isolated spectrin tetramers [28]. While this crude model resembled rotary shadowed EM images, the absence of lateral association between α and β chains along most of their lengths is inconsistent with chemical crosslinking data from spectrin in solution and on intact red cell membranes [29–31]. Further, this concatenation of x-ray crystal structure assumes a gentle left-handed supercoil, while electron microscopy demonstrated that the native quaternary structure of spectrin is a right-handed supercoil [23].…”

“…Chemical crosslinking and crosslink identification were performed as previously described [29,30]. Briefly, LC-MS/MS spectra of tryptic digests from mini-spectrin complexes cross-linked with EDC/sulfo-NHS were compared to those from untreated controls to remove background signals and to facilitate targeted acquisition of high-resolution MS/MS spectra.…”