The common hereditary elliptocytosis-associated α-spectrin L260P mutation perturbs erythrocyte membranes by stabilizing spectrin in the closed dimer conformation

Abstract

• The common HE mutation aL260P reduces spectrin tetramer links between junctional complexes in red cell membranes by favoring closed dimers.• Favoring closed spectrin dimer formation is a new mechanism of red cell membrane destabilization by hereditary anemia mutations.Hereditary elliptocytosis (HE) and hereditary pyropoikilocytosis (HPP) are common disorders of erythrocyte shape primarily because of mutations in spectrin. The most common HE/HPP mutations are located distant from the critical ab-spectrin tetramerization site, yet still interfere with formation of spectrin tetramers and destabilize the membrane by unknown mechanisms. To address this question, we studied the common HE-associated mutation, aL260P, in the context of a fully functional mini-spectrin. The mutation exhibited wild-type tetramer binding in univalent binding assays, but reduced binding affinity in bivalent-binding assays. Biophysical analyses demonstrated the mutation-containing domain was only modestly structurally destabilized and helical content was not significantly changed. Gel filtration analysis of the aL260P mini-spectrin indicated more compact structures for dimers and tetramers compared with wild-type. Chemical crosslinking showed structural changes in the mutant mini-spectrin dimer were primarily restricted to the vicinity of the aL260P mutation and indicated large conformational rearrangements of this region. These data indicate the mutation increased the stability of the closed dimer state, thereby reducing tetramer assembly and resulting in membrane destabilization. These results reveal a novel mechanism of erythrocyte membrane destabilization that could contribute to development of therapeutic interventions for mutations in membrane proteins containing spectrin-type domains associated with inherited disease. (Blood. 2013;122(17): [3045][3046][3047][3048][3049][3050][3051][3052][3053] IntroductionThe hereditary elliptocytosis (HE) syndromes are the most common disorders of erythrocyte shape. In parts of Africa, the incidence of HE approaches that of sickle cell disease, presumably because HE red cells confer some resistance to invasion by malaria parasites. Most cases of HE are due to missense mutations in the erythrocyte membrane skeleton protein spectrin. 1,2 Spectrin is a critical component of the erythrocyte membrane skeleton, a network of proteins on the cytoplasmic face of the membrane that maintains the shape and elasticity of the red cell membrane. 3,4 In the erythrocyte, spectrin exists as an antiparallel ab-heterodimer that self-associates in a head-to-head fashion to form tetramers and higher order oligomers. 5 Studies of spectrin mutations in HE and the related disorder hereditary pyropoikilocytosis (HPP) 6,7 have provided important insights into the structure and function of spectrin in the cell. 8 Furthermore, erythrocyte spectrin has served as the paradigm for our understanding of spectrin proteins in nonerythroid cells. We now know that spectrin mutations in other cell types play important roles in pathogen...

Full-Length Anion Exchanger 1 Structure and Interactions with Ankyrin-1 Determined by Zero Length Crosslinking of Erythrocyte Membranes

“…However, one loop in the crystal structure of the N-terminal domain appears to have a substantially different orientation in the membrane as reflected by crosslink group 3 which had a Cα spacing of 22 Å in the crystal structure (Table 1). This crosslink involved two surface loops so it was not surprising that it was larger than the typical maximum spacing of up to 12 Å for well-ordered regions, but it was also somewhat beyond the typical maximum distance of up to ~16 Å observed for some flexible loop regions and subunit interfaces as reported previously (Harper et al, 2013; Rivera-Santiago et al, 2015a, 2015b; Sriswasdi et al, 2014b). However, we have occasionally observed other cases where zero-length crosslinks identified differences in orientation of surface loops relative to a crystal structure (Rivera-Santiago et al, 2015a).…”

“…The entire stained area of the gel was digested with trypsin including the top of the gel where most of the crosslinked proteins were located, particularly for later time points. Similar to our previous workflows on simpler protein complexes (Harper et al, 2013; Rivera-Santiago et al, 2015b; Sriswasdi et al, 2014a; Sriswasdi et al, 2014b), we used label-free LC-MS/MS comparisons of crosslinked and uncrosslinked controls samples to identify putative crosslinked peptides. However, in this study the increased capability of the Thermo Scientific Q Exactive Plus instrument to collect high-resolution data at high speed eliminated the need for subsequent targeted LC-MS/MS runs (Sriswasdi et al, 2014b).…”

“…To expand the utility of zero-length crosslinking experiments, we recently developed a label-free differential liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and a software package, ZXMiner that was specifically designed and optimized for analyzing zero-length crosslinked samples. We demonstrated the utility of this approach for determining structures that were too large or too flexible for crystallization (Harper et al, 2013; Rivera-Santiago et al, 2015a) or involved conformational changes in solution that could not be readily probed by nuclear magnetic resonance (Sriswasdi et al, 2014a). Furthermore, our recent adaptation of the method to newer, higher duty cycle mass spectrometers has enabled the examination of very large biological systems (Rivera-Santiago et al, 2015b).…”

Clinical Emergencies Presented by Anemia

“…Hereditary elliptocytosis, that is due to defects in either the structure or quantity of the cytoskeletal proteins which is responsible for maintaining the biconcave morphology of RBCs. Mutations in either alpha-and beta-spectrin are most commonly responsible, but mutations in other cytoskeletal proteins (band 4.1 and glycophorin) are also described [11]. Haemoglobin defect: The structural and functional abnormalities to the major oxygen transporting protein of RBC (haemoglobin) can also lead to Haemolytic anaemia.…”

Spectrin: Structure, function and disease

“…Some missense mutations reside in the first six repeats of the spectrin subunit, and the C-terminally truncated  subunit (CpG islands) is a mutational hotspot [56]. The L260P mutation in  spectrin increases the stability of the closed dimer state, perturbing erythrocyte membranes with a fully functional mini-spectrin [57]. All HS have one common feature: the loss of cell membrane surface area causes a spherical phenotype and an increased level of erythrocyte osmotic fragility.…”