Estimation of the secondary structure of protein a from S.aureus by CD-spectroscopy

MolecularImmunology, Printed in Great Britain.

Vol. 19, No. 7, pp. 9S7-959,

1982.

0161-5890/82/070957-03SO3.00/0 Pergamon Press Ltd.

PRELIMINARY COMMUNICATION

ESTIMATION

OF THE SECONDARY

STRUCTURE

OF PROTEIN A FROM S.AUREUS

BY CD-SPECTROSCOPY.

R.Lindmark

Department Present

of Medical

address:

(First

and Physiological Chemistry,Uppsala University,Box 575, S-751 23 Uppsala,Sweden. Sjukhusapoteket,GBllivare Lasarett,S-972 00 Gallivare,Sweden.

received

14 January

1982; accepted

in revised

form 24 March 1982)

Abstract The secondary structure of protein A(SpA) was estimated to 31 % &-helix,13 % p-structure and 56 I random coil by CD-spectroscopy,which is close to the sum of the secondary structures of the SpA fragments.This supports that the SpA molecule is composed N-terminally of four Fc-binding units each consisting of two antiparallel o(-helices interconnected by random coil segments and C-terminally of an region mainly in random coil. Introduction Protein A of S.aureus is located in the cell wall of the bacteria and has the ability to interact with the Fc region of IgG of most mammalian species(Forsgren and SjBquist, 1966;Kronvall et al .,1970) and also with other immunoglobulin classes(Harboe and Fijlling,1974;Johansson and Inganas,l978).This property has made SpA a widely used reagent in immunological methods(Goding,1978). SpA has a molecular mass of 42,000 and exhibits a markedly extended shape(BjBrk et al., 1972).Sequence analysis has revealed a composition of two functionally and structurally different regions(Sjbdahl,l977a).The N-terminal part consists of four consecutively arranged,highly homologous Fc-binding units with a molecular mass of about 7OOO(Sjbdahl,1977b).The C-terminal part of the protein,comprising a molecular mass of 15,000, is a region of extended shape with no Fc-binding ability,but is covalently attached to the cell wall(Sjddahl,l977a). The interaction between the Fc-part of human IgG and an Fc-binding unit of SpA has been binding studied by X-ray crystallography(Deisenhofer et al., 1978;Deisenhofer,l981).The site for SpA on the Fc-part was found to be located mutually on the CH2 and CH domains. Furthermore,the three-dimensional structure of the Fc-binding unit was establi.zhed.The secondary structure of SpA in solution has been studied earlier by circular dichroism (CD)(Sjoholm,l975).This revealed an d-helical content of 48 %,which is much higher than the sum of the W-helical content of the fragments constituting the SpA molecule(Sjodah1, 1977a)This gave the reason to reinvestigate the CD-spectra of SpA,since such a great difference makes it difficult to draw conclusions regarding the structure of SpA from data obtained from its fragments. Materials

and Methods

Cell-bound SpA from the S.aureus strain Cowan l(NCTC 8530) and extracellular SpA from the methicillin resistant strain A 676 were purified as previously described(Hjelm et al.,1972;Lindmark et al.,1977).The SpA was further purified on a Sephadex G-200 column (Pharmacia Fine Chem.AB,Sweden) in 0.001 M phosphate buffer-O.15 M KC1 pH 7.4 and the purity was checked by disc electrophoresis in dodecyl sulphate(Lindmark et a1.,1977). CD spectra were recorded on an automatic JASCO J-41A spectropolarimeter(Japan SpeCtrOscopic Co.,Tokyo) as previously described(Sjodahl,l977a).A rectangular cell with a 12.25 mm path lenght was used and the samples(approx.0.5-0.75 mg/ml),which were in 0.001 M phosphate buffer-O.15 M KC1 pH 7.4,were filtered through a Millipore filter (pore size 0.22 pm).The molar concentrations of SpA were determined both by amino acid analysis on a Beckmgn 121-M amino acid analyzer after hydrolysis in 6 M HCl wit$ 1 % (w/v) phenol at 110 C for 24 h,and from the UV spectra of the solutions with A275=1.46 (Lindmark et a1,1977).The ellipticity[@Jas calculated on a mean residue weight basis.The secondary structure components were estimated from a mean value Of two 951

R. LINDMARK

958

different spectra of two different field and Fasman,1969.

SpA preparations

according

to the method

of Green-

Results and discussion. The secondary structure of cell-bound SpA from strain Cowan 1 has previously been estimated from the CD spectra as:oc-helix 48 %,B_structure 21 % and random coil 31 %(Sjoholm,l975).When the secondary structure calculated from the CD spectra of the SpA fragments D,A,B and CX,together constituting the entire SpA molecule(Sjddahl,l977a),were added an&-helical content of 29 % was obtained(Table l).This implies that the fragmentation procedure causes a loss ofcx-helical structure by 19 %,which,further,represents a 30 % loss of=-helix in the Fc-binding units.This would in fact indicate a nearly completeK-helical structure in the regions interconnecting the Fc-binding units,which is turned into random coil by the proteolytic cleavage.This is in contradiction to the findings in an NMR study of intact SpA,which suggests the excistence of hydrophilic, flexible regions between the Fc-binding units(Wright et al.,1977).These observations gave the reason to reinvestigate the CD spectra of SpA in the far UV. Table 1. Calculated

percentages

ofo!-helix&structure

Percentage M-helix

Source

SpA Cowan 1

and random coil in SpA.

of p-structure

random coil

(a)

(b)

(a)

(b)

(a)

(b)

31

ND

13

ND

56

ND

SpA A 676

29

ND

12

IJD

59

ND

SpA Cowan 1'

48

55

21

13

31

32

fragmented

29

27

18

24

53

49

SpAd

a/ b/ c/ d/

calculated according to Greenfield and Fasman,1969. calculated accordinn to Chen et a1.,1972. from Sjoholm,1975. _ the sum of the secondary structure of SpA fragments D,A,B and CX,together comprising the SPA molecule(Sjiddahl,l977a).The previouslv estimated structure of each fraament from CD spectra was utilized to calculate the"number of residues in each structure. The number of residues for each structure i.e. o&helix&structure and random coil, were added,and from the total number of residues the percentage secondary structure could be calculated. ND=Not determined.

The CD spectra of SpA from the Cowan 1 strain,which represents cell-wall-bound SpA,and extracellular SpA from a methicillin resistant strain A 676 are shown in Figure l.The two different SpA:s of different sources exhibit great spectral similarities and in addition the secondary structure is identical within the experimental error(Table 1). This is in accordance with previously noted similarities in physicochemical properties, amino acid compositions and affinity constants for IgG between the two forms of SpA (Lindmark et al.1977;Lindmark et al.l981).The a-helix and random coil contents of SpA Cowan 1 were 31 % and 56 %,respectively(Table l),which differ from previously obtained values of 48 % and 31 %(Sjoholm,l975).This difference is explained by dissimilarities in the purification methods employed,and moreover,in earlier studies on SpA a higher specific absorbance was frequently used.The M-helical content of a Fc-binding unit, fragment B,have been calculated to 47 % by CD(Sjtidahl,l977a) and to 36 % by X-ray crystallography(Deisenhofer,l981).This difference is not remarkable since an over estimation of the M-helical content by CD measurements is often obtained(Nystrom,l980). Moreover,the X-ray crystallography was performed at pH 4.1 and fragment B was complexed with the Fc part of IgG.No p-structure was found in fragment B by X-ray crystallography in contrast to the 15 %p-structure calculated from the CD spectra.This indicates that the only ordered structure in the Fc-binding region of SpA would be &-helix. The values of the secondary structure of SpA in this report are very close to those calculated for fragmented SpA(Table l).This indicates that no gross conformational changes are introduced in the fragments by the proteolytical cleavage.The similarities in secondary structure between SpA and fragmented SpA revealed by the CD spectra make it possible,together with the known three dimensional structure of one Fc-binding unit (Deisenhofer,l98l),to propose the over-all structure of SpA:The four Fc-binding units, consisting of two antiparallel o(-helices,are connected to each other by random coil segments and the C-terminal part of the molecule,the X-part,consists of a very extended region consisting mainly of random coil.This picture of SpA with globular,Fc-binding units interconnected by flexible random coil segments is in agreement with that obtained in the NMR study of SpA(Wright et a1.,1977).

959

Secondary Structure of Protein A from S. Aureus

WAVELENGTH 210

240

230

220

Figure 1. The CD spectra of SpA from the Cowan 1 strain(-O-)and of extracellular SpA of strain A 676(-A-) in 0.001 M potassium phosphate buffer-O.15 M KC1 pH 7.4. Acknowledgement This work was supported by the Swedish Medical Research Council(project no.l3X-2518). Dr P.Edman is thanked for valuable advice concerning the recording of the CD spectra and Professor J.Sjdquist for discussion. References Bjork,I.,Petersson,B-A.and Chen,Y.H.,Yang,J.T.

Sjoquist,J.(1972)

and Martinez,H.M.(1972)

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661

Z.

NM