Conformational Stabilities of the Structural Repeats of Erythroid Spectrin and Their Functional Implications

Abstract

The two polypeptide chains of the erythroid spectrin heterodimer contain between them 36 structural repeating modules, which can function as independently folding units. We have expressed all 36 and determined their thermal stabilities. These vary widely, with unfolding transition mid-points (Tm) ranging from 21 to 72 °C. Eight of the isolated repeats are largely unfolded at physiological temperature. Constructs comprising two or more adjacent repeats show inter-repeat coupling with coupling free energies of several kcal mol-1. Constructs comprising five successive repeats from the β-chain displayed cooperativity and strong temperature dependence in forced unfolding by atomic force microscopy. Analysis of aligned sequences and molecular modeling suggests that high stability is conferred by large hydrophobic side chains at position e of the heptad hydrophobic repeats in the first helix of the three-helix bundle that makes up each repeat. This inference was borne out by the properties of mutants in which the critical residues have been replaced. The marginal stability of the tertiary structure at several points in the spectrin chains is moderated by energetic coupling with adjoining structural elements but may be expected to permit adaptation of the membrane to the large distortions that the red cell experiences in the circulation. The two polypeptide chains of the erythroid spectrin heterodimer contain between them 36 structural repeating modules, which can function as independently folding units. We have expressed all 36 and determined their thermal stabilities. These vary widely, with unfolding transition mid-points (Tm) ranging from 21 to 72 °C. Eight of the isolated repeats are largely unfolded at physiological temperature. Constructs comprising two or more adjacent repeats show inter-repeat coupling with coupling free energies of several kcal mol-1. Constructs comprising five successive repeats from the β-chain displayed cooperativity and strong temperature dependence in forced unfolding by atomic force microscopy. Analysis of aligned sequences and molecular modeling suggests that high stability is conferred by large hydrophobic side chains at position e of the heptad hydrophobic repeats in the first helix of the three-helix bundle that makes up each repeat. This inference was borne out by the properties of mutants in which the critical residues have been replaced. The marginal stability of the tertiary structure at several points in the spectrin chains is moderated by energetic coupling with adjoining structural elements but may be expected to permit adaptation of the membrane to the large distortions that the red cell experiences in the circulation. Spectrin, an elongated flexible tetrameric molecule, defines the unusual shear resistance and elasticity of the red cell membrane (1Mohandas N. Evans E. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 787-818Crossref PubMed Scopus (458) Google Scholar, 2Discher D.E. Carl P. Cell Mol. Biol. Lett. 2001; 6: 593-606PubMed Google Scholar). The tetramers are made up of two αIβI heterodimers, associated head-to-head (3Shotton D.M. Burke B. Branton D. J. Mol. Biol. 1979; 131: 302-329Crossref Scopus (368) Google Scholar). Both the α and the β chain are characterized by a series of homologous repeating units of about 106 amino acids, each with the structure of a loose triple-stranded α-helical coiled-coil (4Yan Y. Winograd E. Viel A. Cronin T. Harrison S.C. Branton D. Science. 1993; 262: 2027-2030Crossref PubMed Scopus (336) Google Scholar, 5Speicher D.W. Marchesi V.T. Nature. 1984; 311: 177-180Crossref PubMed Scopus (307) Google Scholar). Repeats of this nature also characterize the many proteins that comprise the spectrin superfamily (6Muse S.V. Clark A.G. Thomas G.H. J. Mol. Evol. 1997; 44: 492-500Crossref PubMed Scopus (11) Google Scholar, 7Baines A.J. Cell Mol. Biol. Lett. 2003; 8: 195-214PubMed Google Scholar, 8Djinovic-Carugo K. Young P. Gautel M. Saraste M. Cell. 1999; 98: 537-546Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 9Grum V.L. Li D. MacDonald R.I. Mondragon A. Cell. 1999; 98: 523-535Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). Successive spectrin repeats are joined by a continuous α-helix, of which the N-terminal half forms the C-terminal helix (C-helix) of the three-helix bundle of one repeat and the other half forms the first helix (A-helix) of the adjoining repeat; 5 residues in the center of the helix are not part of the repeating structure and are referred to as a “linker” region (9Grum V.L. Li D. MacDonald R.I. Mondragon A. Cell. 1999; 98: 523-535Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 10Kusunoki H. Minasov G. Macdonald R.I. Mondragon A. J. Mol. Biol. 2004; 344: 495-511Crossref PubMed Scopus (89) Google Scholar, 11Ortiz V. Nielsen S.O. Klein M.L. Discher D.E. J. Mol. Biol. 2005; 349: 638-647Crossref PubMed Scopus (75) Google Scholar). It has been suggested (10Kusunoki H. Minasov G. Macdonald R.I. Mondragon A. J. Mol. Biol. 2004; 344: 495-511Crossref PubMed Scopus (89) Google Scholar, 11Ortiz V. Nielsen S.O. Klein M.L. Discher D.E. J. Mol. Biol. 2005; 349: 638-647Crossref PubMed Scopus (75) Google Scholar) that the flexibility of the spectrin chain under physiological conditions may be governed by kinks resulting from a low conformational stability of some of the linkers. On the other hand, the folding of individual repeats is cooperative, which implies that thermal “melting” of a linker would cause the repeats on either side to unfold. It has indeed been found that some isolated repeats have only marginal stability and are partly or largely unfolded at 37 °C (12Kusunoki H. MacDonald R.I. Mondragon A. Structure (Camb.). 2004; 12: 645-656Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 13MacDonald R.I. Cummings J.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1502-1507Crossref PubMed Scopus (38) Google Scholar). At the same time, the thermal, solvent-induced, or forced unfolding of constructs containing contiguous repeats has been found in some cases to display a conformational coupling between them (14MacDonald R.I. Pozharski E.V. Biochemistry. 2001; 40: 3974-3984Crossref PubMed Scopus (46) Google Scholar). To establish how repeats of low stability are distributed through the spectrin molecule, we have examined the thermal unfolding of all of the 36 repeats that make up the two chains of erythroid spectrin. Some of the repeats show exceptionally high stability, the structural origins of which we have tried to identify. We have additionally compared thermal unfolding profiles of single repeats with those of several two-repeat fragments and with two five-repeat constructs to investigate the extent of conformational coupling within the chains. Where discrete unfolding transitions of contiguous repeats can be observed, analysis of the propagation of conformational cooperativity along the polypeptide chain becomes possible. Implications for the mechanical function of spectrin in the cell are considered. Materials—pGEX-4T-2 vector and glutathione-Sepharose 4B were purchased from Amersham Biosciences. pET-31b(+) vector and nickel resin were from Novagen (Madison, WI). QuikChange site-directed mutagenesis kit and BL21 (DE3) bacteria were from Stratagene (La Jolla, CA). Restriction enzymes were from New England BioLabs (Beverly, MA). Proteinase inhibitor mixture set II was from Calbiochem. Reduced form glutathione, thrombin, isopropyl-1-thio-β-d-galactopyranoside, and bovine serum albumin were from Sigma. SDS-PAGE and electrophoresis reagents were from Bio-Rad, and GelCode staining reagent was from Pierce. All other chemicals were reagent grade from standard sources. Design and Subcloning of Recombinant Spectrin Polypeptides—The boundaries of all repeats were defined by the SMART data base, with two exceptions; β-spectrin repeat 1 (here designated βIR1+) starts 8 residues before the predicted repetitive segment (15Ursitti J.A. Kotula L. DeSilva T.M. Curtis P.J. Speicher D.W. J. Biol. Chem. 1996; 271: 6636-6644Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), whereas α-spectrin fragment 9-10 contains a triple helix βIR9 interrupted by an SH3 domain (segment 10) between helices B and C. We expressed these as a single polypeptide and did not attempt to make a separate construct of βIR9 without the SH3 domain. To ensure proper folding, we extended the N and C termini of our single-repeat constructs by 5 residues. The residue numbers of all sequences are given in Table 1. αIR14, αIR18, and αIR19 were subcloned into the pGEX-4T-2 vector, using BamHI and SalI. All other spectrin single repeats and two-repeat fragments were subcloned into the pGEX-4T-2 vector, using EcoRI and SalI restriction enzymes upstream and downstream, respectively. αIR18 was also subcloned into the pET-31b(+) vector, using NsiI and XhoI upstream and downstream, respectively. One five-repeat fragment βIR5-9 was subcloned as described previously (16An X. Guo X. Sum H. Morrow J. Gratzer W. Mohandas N. Biochemistry. 2004; 43: 310-315Crossref PubMed Scopus (100) Google Scholar), and another five-repeat fragment βIR6-10 was subcloned into the pGEX-4T-2 vector, using EcoRI and SalI restriction enzymes. The template used to amplify α-spectrin single repeats and tandem-repeat fragments was full-length α-spectrin cDNA. Similarly, full-length β-spectrin cDNA was used to amplify β-spectrin single repeats and tandem-repeat fragments. Mutations found in αIR14, αIR15, βIR3, and βIR7 constructs were corrected by site-directed mutagenesis using the Stratagene QuikChange kit. The fidelity of all the constructs was confirmed by DNA sequencing. Mutations were incorporated into repeat sequences using the Stratagene QuikChange kit and verified by sequencing.TABLE 1Information of erythrocyte spectrin single repeatsNameAmino acidsNucleotidesNameAmino acidsNucleotidesαIR145-163133-489βIR1+293-422877-1266αIR2154-269460-807βIR2426-5321276-1596αIR3260-375778-1125βIR3523-6411567-1923αIR4365-4811093-1443βIR4632-7471894-2241αIR5472-5871414-1761βIR5738-8522212-2556αIR6578-6921732-2076βIR6843-9582527-2874αIR7683-7982047-2394βIR7949-10652845-3195αIR8789-9022365-2706βIR81056-11713166-3513αIR9-10893-10872677-3261βIR91162-12783484-3834αIR111078-11883232-3564βIR101269-13823805-4149αIR121179-12943535-3882βIR111374-14724120-4446αIR131285-14003853-4200βIR121473-15884417-4764αIR141391-15054171-4515βIR131579-16944735-5082αIR151496-16114486-4833βIR141685-18015053-5403αIR161602-17174804-5151βIR151792-19075374-5721αIR171708-18225122-5466βIR161898-20135692-6039αIR181813-19325437-5796αIR191923-20475767-6141αIR202038-21556112-6465αIR212146-22646436-6792 Open table in a new tab Preparation of Recombinant Spectrin Polypeptides—The cDNA encoding the desired polypeptide was transformed into Escherichia coli BL21. Because some of the polypeptides were insoluble when expressed at 37 °C, all the fragments were expressed at 16 °C. This greatly improved the yield of soluble product and ensured that all the polypeptides were folded under the same conditions. All the polypeptides were satisfactorily expressed and soluble, although the yields varied from 1 to 50 mg/500 ml bacterial culture. Expression of recombinant proteins was induced by 0.1 mm isopropyl-β-d-thiogalactopyranoside at 16 °C for 3-4 h. Glutathione S-transferase fusion polypeptides were purified on a glutathione-Sepharose 4B affinity column. Glutathione S-transferase was cleaved with thrombin. The exception was αIR18, which could not be recovered after thrombin cleavage and was therefore expressed in His-tagged form and purified on a nickel column. Concentrations of most polypeptides were determined spectrophotometrically, using extinction coefficients calculated from the tryptophan and tyrosine contents (17Perkins S.J. Eur. J. Biochem. 1986; 157: 169-180Crossref PubMed Scopus (546) Google Scholar). βIR9 does not contain tryptophan and βIR11 does not contain tyrosine, and because of their low absorbances, an approximate concentration of these two repeats was obtained by the Bradford (18Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216033) Google Scholar) method, with bovine serum albumin as standard. Proteins were dialyzed against phosphate-buffered saline and clarified before use by ultracentrifugation at 230,000 × gav for 30 min at 4 °C. Mass Spectrometric Analyses—Mass spectrometric analyses were performed in an Applied Biosystems (Applied Biosystems, Foster City, CA) Voyager DE MALDI mass spectrometer. Samples at a concentration of 10 μm were mixed with an equal volume of sinapinic acid matrix solution in 50% acetonitrile, 0.1% trifluoroacetic acid (Sigma), and 1-2 μl of each mixture was spotted onto a sample plate and dried. The results were externally calibrated against aldolase (Sigma; [M+H]+= 39, 212.28) and bovine serum albumin ([M+H]+= 66,430.09). Sedimentation Equilibrium Analysis—The molecular weights of proteins were measured by sedimentation equilibrium in a Beckman Coulter ProteomeLab™ XL-A analytical ultracentrifuge at a rotor speed of 20,000 rpm. Equilibrium distributions were scanned at 280 and 235 nm. Partial specific volumes were calculated from amino acid compositions. Circular Dichroism Spectra and Thermal Unfolding—Far-UV CD spectra were recorded in a Jasco 700 spectropolarimeter, equipped with a thermostated cell housing, in cells of 1-mm path length. For determination of unfolding profiles, the temperature was increased from 4 to 90 °C at a rate of 0.4 °C/min, and the ellipticity was recorded at 2o intervals. To test for reversibility, several of the were to 4 °C, and the CD was To unfolding from the ellipticity temperature profiles, were made for the in ellipticity in the temperature and the transition individual residues to the and e in the heptad hydrophobic we aligned all the sequences with fragment for which the heptad repeats have been defined (9Grum V.L. Li D. MacDonald R.I. Mondragon A. Cell. 1999; 98: 523-535Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 10Kusunoki H. Minasov G. Macdonald R.I. Mondragon A. J. Mol. Biol. 2004; 344: 495-511Crossref PubMed Scopus (89) Google Scholar), with the of C. J. J. Mol. Biol. PubMed Scopus Google Scholar). For of of individual or we used the N. 1997; PubMed Scopus Google Scholar). were displayed and in of side chains was using D. P. 23: PubMed Scopus Google Scholar). by used atomic force microscopy. were made on βIR5-9 and βIR6-10 using described and analyses G. S. G. Speicher D.W. Discher D.E. Biophys. J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). a CA) was equipped with a cell and a to and temperature. For one were and with the of a analysis in unfolding are and the is in many and of is to an of the unfolding Design and of Recombinant Spectrin spectrin fragments examined are defined in Table 1. All contain an residues from the vector at the N All the expected chain and as measured by mass Sedimentation equilibrium each of the to be and Circular spectra of all expressed fragments the of proteins of high with the exception only of which contains the SH3 domain and therefore has a from the residue ellipticity at N. Biochemistry. 8: PubMed Scopus Google Scholar) as the thermal unfolding transitions also all fragments the folded at low temperature. of Recombinant Spectrin thermal transition profiles of single repeats of all we a of a unfolding with the of all individual α-spectrin and C and display those of the β-chain For all polypeptides the transition was more or in ellipticity and the region of as also by in the we have a to the of tertiary structure unfolded at each temperature. results show that the thermal of the individual repeats from spectrin chains widely, with transition mid-points (Tm) ranging from 21 and 72 °C B and the 36 repeats up the two 8 have 37 °C. of of of unfolding data for all 36 erythroid spectrin repeats to for structural that the of stabilities. have to between the tryptophan through most of the repeats (12Kusunoki H. MacDonald R.I. Mondragon A. Structure (Camb.). 2004; 12: 645-656Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, R.I. A. Saraste M. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar), as a one stability has been to the of in helix B of the three-helix bundle (12Kusunoki H. MacDonald R.I. Mondragon A. Structure (Camb.). 2004; 12: 645-656Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). We have on the exceptionally the of the is by This is not a for is found also in repeat 36 could we in our between stability and the of the residues at a and of the heptad hydrophobic repeat. On the other hand, was a in repeats at other in the this was when the data were with analyses of and At the and e and in helix the most repeats have at 1 hydrophobic about half the helix and have residues are or at and at the these are by and modeling of that helix residues and are to residues and in helix C and may form a hydrophobic the repeat (12Kusunoki H. MacDonald R.I. Mondragon A. Structure (Camb.). 2004; 12: 645-656Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), residues at and in helix are to in helix Mutations of the and at and in as would the hydrophobic are therefore predicted to the We the mutants and and the At low the of the and polypeptides were that the side chains are with folding of the triple the of these on their thermal stabilities. The single the of 72 °C in the to °C in and °C in The has °C, the for all The free energies of to the two single and the at the of the polypeptide are about and kcal mol-1. be an residue in the and we that indeed at these in a an we acid because this is the residue in these and with high °C in as has of °C, with a free of at the of about kcal mol-1. the acid is more the but acid in the stability by and in the We that a high of the side chains in these may for the high of the repeat. of structural of contiguous repeats are we each of two and and show the thermal profiles of and with the calculated profiles for the It can be that in is of the by the more folding the of the temperature transition is is at this to an unfolded segment of polypeptide The of to coupling with the of in in and βIR9 in and °C. The corrected profiles the of the equilibrium for unfolding at each temperature within the transition α is the unfolding at the given and from a of the free of a repeat at the of the repeat with the that The in free of unfolding is also by the of and J.A. PubMed Scopus Google Scholar), as × is the of unfolding and is the in This that is the same for fragments and that within the temperature in can be as We found conditions to be and the two that the free energies of at the transition of by of by and of β by and kcal mol-1. The nature of conformational coupling between the repeats along the spectrin chains is most expressed by a of the unfolding profiles of the single repeats with that of the chains. We show only the for the the data for the and are to that conformational from between the chains. It is at that is unfolding in part of the spectrin at physiological temperature. The of the that unfolding within a temperature at about 50 °C. This is in with the of of in solution and on the which a at °C L. K. Biochemistry. PubMed Scopus Google this is also the temperature at which the red cell membrane Mohandas N. J. PubMed Scopus Google Scholar). of in a fragment of five successive was also We of this fragment to in more the of 4 two in the unfolding by transition with a to °C and for about of the in the unfolding of We also the unfolding by in forced unfolding using to the of folded units at At °C, up to five unfolding were in as the construct was to forced unfolding for This that up to five structural at low temperature. At 30 °C, only transitions were in only which suggests of of is between 30 and °C, with of and °C temperature with our of was To the may be by with the adjacent repeat on the C-terminal side in the β-spectrin we also the construct This to to βIR5-9 in of thermal and forced unfolding We have defined the conformational stability along the and the β-chain of the erythroid The of in the two associated chains of the by MacDonald and Cummings R.I. Cummings J.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1502-1507Crossref PubMed Scopus (38) Google Scholar), is not a when the chains are the marginal stability of some repeats at points along the chains is a of the It can be expected to for the large membrane induced by shear many other on thermal, solvent-induced, and mechanical unfolding (10Kusunoki H. Minasov G. Macdonald R.I. Mondragon A. J. Mol. Biol. 2004; 344: 495-511Crossref PubMed Scopus (89) Google Scholar, R.I. Pozharski E.V. Biochemistry. 2001; 40: 3974-3984Crossref PubMed Scopus (46) Google Scholar, N. T. D. N. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, S. A. J. J. Mol. Biol. 2005; 349: PubMed Scopus Google Scholar, Carl P. S. P. Speicher D.W. D.E. Biophys. J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), have contiguous spectrin repeats may be We have the coupling free energies between of and we have that coupling within a fragment of five successive It that through of successive repeats is along the two chains and that the of some isolated repeats at physiological temperature is by this also be that for a more of the stability of the spectrin the of temperature on inter-repeat coupling be into S. A. J. J. Mol. Biol. 2005; 349: PubMed Scopus Google Scholar) found a of cooperativity in unfolding with but the is the in the forced unfolding transitions Some into the the stability of the spectrin repeats results from an of We have hydrophobic and at the e in the heptad repeats of helix that are from suggests that form a hydrophobic with residues and in the helix C. The of these residues to be critical for function in all sequences from to the to and are by or is or only to in all from to 16 of the adjacent repeat with the α-spectrin N G. D. Gratzer Eur. Biophys. J. 1999; PubMed Scopus Google Scholar), and the stability of may therefore be by the are in that could test this The on βIR5-9 and βIR6-10 that by about °C, repeats 8 and of β-spectrin are of mechanical CD that most this temperature. spectrin in we may that these repeats an extended under mechanical The the of unfolding of spectrin repeats in solution at physiological and their stability with to mechanical unfolding to tetramers in the of the red We have not found of thermal profiles of repeats X. and N. in the of the of spectrin is the same in red cell as in solution L. K. Biochemistry. PubMed Scopus Google Scholar) also for the of solution data to physiological the cells of as spectrin is to membrane against from and to at M. J. Cell Biol. PubMed Scopus Google Scholar, S. L. V. J. Cell Biol. PubMed Scopus Google Scholar). It that as the be more to mechanical and this may in high thermal stability of the Spectrin isolated from is erythrocyte spectrin J. V. J. Biol. Chem. Full Text PDF PubMed Google Scholar). spectrin is a mixture of but the of a structure more to erythrocyte are to and how the of is to the conformational stability of