Effects of Calcium, Magnesium, and Phosphorylcholine on Secondary ...

THEJOURNAL OF BIOLCGICAL Cmwslllv

Vol. 269, No. 9, Issue of March 4, pp. 6424-6430, 1994 Printed in U.S.A.

0 1994 by The American 90ciety for Biochemistry and Molecular Biology, Inc.

Effects of Calcium, Magnesium, and Phosphorylcholine on Secondary Structuresof Human C-reactive Protein and Serum Amyloid P Component Observed by Infrared Spectroscopy* (Received forpublication, October 14,

1993, and in revised form, November 15,

1993)

Aichun Dong*, WinslowS . CaugheySO, and Terry W. Du Closen From the $Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523 and the Wepartment of Medicine, Veterans Administration Medical Center and University of New Mexico, Albuquerque, New Mexico 87108

The secondary structures of human C-reactive protein tity is shared by mouse CRP and human CRP (5, 6). Human (CRP) and serum amyloid P component ( S A P ) in D,O- CRP and S A P share 50 and 72% sequence homology with hambased solutions in the presence or absence of calcium, ster FP, respectively (7).The evolutionary conservation of penmagnesium, and phosphorylcholine have been investi- traxin genes and ubiquitous presence of pentraxins in vertegated using Fourier transform infrared spectroscopy. bratessuggest a n essential function for these proteins. 50% Although a compelling reason for the existence of these proQuantitative analysis provided estimations of about @-sheet,12% a-helix, 24% @-turn,and 14% unordered teins has not been found, all pentraxins haveCa2+-dependent structure for CRP and about 54% @-sheet,12%a-helix, binding activities and many are acute phase reactants(8-15). 25% @-turn, and9% unordered structure for SAP. With CRP andFP have a primary bindingspecificity for phosphorylbothproteinssignificantcalcium-dependentchanges choline (PC) which is absent from SAP. Human CRP andS A P wereobserved in conformation-sensitiveamide I regions assigned to each type of structure. The CRPspec- and hamster FP all activate complement when bound to liCRP has been demonstrated to bind substrates trum was also af€ectedby magnesium, but the changes gands (16, 17). (12, 14),histones (14, 15), andsmall including chromatin differed from those inducedby calcium. The S A P specnuclear ribonucleoproteins (13). These binding reactions are trum was not affected by magnesium. Phosphorylchospecific for the protein moiety and are inhibited by free PC. line in the presence of calcium also affected the spectrum of CRP but not the spectrum of SAP. Our present High aftinity chromatin binding activity was also observed in S A P (18).SAP and FP have been identified as peripheral comstudy provides the first direct comparison of the secondary structures of the pentraxins human CRP and S A P ponents of a variety of amyloids (19-21), and it appears that andhamsterfemaleprotein(Dong, A., Caughey, B., the levels of FP expression can have a profound influence on Caughey, W. S., Bhat, K. S., and Coe,J. E.(1992)Biochem- amyloid formation in vivo (22). There have been few reports on the secondary structure of istry 32,9364-9370).Thesefindingssuggestthatthe three pentraxins have similar secondary structure compentraxins. Knowledge on the secondary structure of pentraxpositions and calcium-dependent conformational ins had beenlimited to a circular dichroism(CD) study of changes, but differ significantly in their responses to human CRP and S A P (23)and an infrared (IR) study of hamphosphorylcholine and magnesium. Such properties are ster FP (24)until a recent abstract reporting x-ray crystalloexpected tobe relevant to the incompletely understood graphic analysis of human SAP (25).About 45% P-sheet and roles of these highly conserved proteins including bind34% a-helix were estimatedfor human CRP and SAP by Young ing to nuclear proteins, complement activation, and asand Williams (23)using CD. About 50% p-sheet, 10% a-helix, sociation with amyloids. 28% p-turn, and10% unordered, a structure often referred toas random coil,2were estimatedfor hamster FP by Dong et al. (24) using IR. X-ray crystallographic analysis shows that the subC-reactive protein (CRP),’ serum amyloid P component unit of human S A P is dominated by an antiparallel p-sheet (SAP),and female protein (FP) belong to a family of proteins structure and containsa single short a-helix which lies above called pentraxins (1,2).Pentraxins, namedfor their cyclic con- the disulfide bridge C ~ s ~ ~ - and C y adjacent s ~ ~ to the glycosylafiguration of five noncovalently bound identical subunits (11, tion site, Asn32 (25). The antiparallel p-sheet structure arare ancient proteins which have evolved at a conservative rate. rangement of the S A P subunit closely resembles concanavalin Limulus CRP isolated from the hemolymph of the horseshoe A and pealectin, despite theabsence of any sequence homology crab shares about 25% amino acid sequence identity with its between these proteins and the pentraxin(25). mammalian counterparts (3,4),whereas 71% sequence idenIn the present study, we examined the secondary structure and the Ca2+- and PC-dependent conformational changes of * This work was supported in part by a giR from Strohtech, Inc. (to human CRP andS A P in solution using IR. To avoid the aggreW. S. C) and Colorado Agricultural Experiment Station Project 643 (to gation found at the high concentrations(215 mg/ml) required W. S. C) and by The Department of Veterans Af€airs and National Institutes of Health Grant A128358 (to T. W. D.). The costs of publication for obtaining a high quality infrared spectrum of protein in SAP were studied in DzOof this article were defrayed in part by the payment of page charges. HzO-based solution,human CRP and This article must therefore be hereby marked “advertisement”in ac- based solutions in this study. Fourier transform IR spectroscordance with 18 U.S.C. Section 1734 solely to indicate this fact. copy, aided by powerful spectral handling techniques, namely !j To whom correspondence should be addressed. Tel.: 303-491-6443; Fax: 303-491-0494(W. S. C.);Tel.: 505-256-5717;Fax: 505-256-2877 Fourier self-deconvolution (enhancement) and second-deriva(T. W. D.). Mooreand Fasman (67)have proposed recently use of the term The abbreviations used are: CRP, C-reactive protein; SAP, serum amyloid P component; FP, female protein; PC, phosphorylcholine; IR, “poly-proline 11” to describe the short, flexible segments often referred to as “random coil.” infrared.

64 24

6425

Effects of Ca2+,Mg2’, and PC on Pentraxins

tive analysis, has become a widely used method for studying I I I I I I the secondary structure of polypeptides and proteins (26-29). Amide I’(1) I1 11’ The amideI band (from 1700 to 1620 cm-’), which is due almost m m W entirely to the C=O stretching vibration of the peptide linkages 4 that constitute the backbone structure, is known to be sensitive * to small variations inmolecular geometry and hydrogen bonding patterns of proteins and hasbeen extensively used in the studies of secondary structure and conformational changes of .. m proteins (27-31). Empirical studies have demonstrated that W the infrared amide I band areas associated with a particular secondary structure (e.g., a-helix, p-sheet, turn, and unordered structures), divided by the sum of all band areas assigned to secondary structures, provide a quantitative estimationof the relative amountsof the secondary structure components which are close to those derived from x-ray diffraction data (27, 28, 3234). The water spectral handling technique developed in our laboratory (35)enables us to obtain infrared spectra of proteins in H20- as well as DzO-based solutions with high quality and reproducibility. Results of our present studies indicate that humanCRP and S A P are predominantly composed I I I I I I 10 1700 1400 1: 0 of antiparallel p-sheet structure, with small amounts of a-helix and unordered structures. Comparative infrared spectra analWAVENUMBER (cm-1) ysis of human CRP, SAP, and hamster FP provide direct eviFIG.1. Infrared spectra of Ca2+-depleted human CRP andSAP dence that pentraxins have similar secondary structure folding in D,O-based solutions and Cas+-depleted hamster FF’in HzOpatterns.Ca2+-dependent conformationalchangeswere de- based solution.The spectra of human CRP and SAP were measured tected in amide I regions assigned to p-sheet, a-helix, p-turn, afier 24-h hydrogen-deuterium exchange at room temperature. The and unordered structure, especially the p-sheet region. The spectral contributions of the buffer and atmospheric water vapor have protein conformational changes induced by binding of M$+ and been subtracted from the original spectra as described under “Materials and Methods.” Human CRP and SAP were at concentrations of 5 mg/ml PC were also detected in CRP. in 20 m~ Tris, 100 m~ NaCl (pD8).The spectrum of hamster FP is from v) v)

d

MATERIALS AND METHODS Purification and Preparation of CRP and SAP-Human CRP was isolated and purified from human pleural fluid and plasmapheresis samples by affinity chromatography, gel filtration, and ion exchange chromatography as described previously(15). Human SAP was isolated and purified from human pleural fluid and plasmapheresis samples as described elsewhere (36). The Ca2+-depletedCRP and SAP in D,O (99.9%, Cambridge Isotope Laboratories, Cambridge, M A ) buffer were obtained by addition of 2 ml of D,O-based buffer with EGTA (20 m~ Tris, 100 m~ NaCl, 2 m~ EGTA, pD 8)to 0.5 ml of CRP (2.9 mg/ml) or 0.5ml of S A P (2.2 mg/ml)and then by concentration of the samples to about 5 mg/ml by centrifugation using a Centricon 10 microconcentrator (Amicon) at 4000 x g. The concentrate was diluted with D,O-based buffer, and the mixture was concentrated as before, a procedure which wasrepeated four times in 24 h. To remove excess EGTA, the D,O-based buffer without EGTA was used in the last cycle of hydrogen-deuterium exchange. The final concentrations of CRP and SAP were about 5 mg/ml. The CRP and SAP samples containing 2 m~ Ca2+,2 m~ Mg”, or 1m~ PC were obtained by adding concentrated stock solutions of CaCI,, MgCI,, or PC to the Ca2+depleted protein solutions. Infrared Measurements and Amide I Spectra Analysis-Protein solutions were prepared for infrared measurement in a CaF, cell (Beckman FH-01) with a 50-pm spacer. Infrared spectra were recorded at 20 “Cwith a Perkin-Elmer model 1800 Fourier transform infrared spectrometer equipped with a Hg/Cd!Ik detector and interfaced with a Perkin-Elmer 7700 computer. For each spectrum a 1000-scan interferogram was collected in single beam mode with a 2cm” resolution and a 1 cm” interval. Reference spectra were recorded under identical scan conditions with only buffer in the cell. Protein spectra were subtracted according to previously established criteria (33) with a double-subtraction procedure (35). The factoring of water vapor subtraction was based on the elimination of water vapor bands outside the amide I region between 1850 and 1720 cm” which resulted in a featureless spectrum in this region of the second-derivativespectrum. The final protein difference spectra after double-subtraction were smoothed with a 9-point Savitsky-Golayfunction (37)to removethe possible white noise. Fourier self-deconvolutions were camed out using a half-bandwidth (Au,,, or full width at half-height) of 16 cm” and an enhance factor ( K value) of 2.4 with the ENHANCE function of a Perkin-Elmer 7700 data station. The second derivative was obtained with a Savitsky-Golay derivative function for a five-data point window. Secondary structure composition was determined by the second-derivative/cue-fitting analysis using

Dong et al. (24). The amide I’ and 11’ bands of proteins in D,O-based solution correspond to the amide I and I1 bands of proteins in HzObased solution. Spectra Calc Software (Galactic Industries Corp.) on a 386-based personal computer. The inverted second-derivativespectra were obtained by factoring by -1 and were fitted with Gaussian band profiles (35). RESULTS

Original Infrared Spectra of Human CRP and SAP-Fig. 1 presents the original infrared spectra of Ca2+-depletedhuman CRP and SAP in D20-based solutions. For comparison, the figure also includesthe spectrumof hamster FP in HzO-based solution as obtained previously (35).The similarity between the amide I’ and 11’ bands of CRP and SAP suggests that the secondary structure compositions of the two proteins are very much alike. After 24 h of hydrogen-deuterium exchange, the spectra of both CRP and SAP exhibit absorbance maxima for their amide I’ and amide 11’ bands at 1633 and 1455 cm-l, respectively. The amideI band absorbance maximum for FP in H20-based solution is at nearly the samewave number as are the amideI’ maxima for CRP and SAP in D20-basedsolutions. Hydrogen-deuterium exchange is found to have only small effects on amide I band frequencies (27).In contrast, amide I1 bands, which arise mainly from a n out-of-phase combination of N-H in-plane bending and C-N stretch vibrations of peptide linkages (311,are typically red-shifted by about 100 cm-l due to exchange of hydrogen by deuterium. Therefore, the 95 cm-I lower wave number of the amide11‘bands for CRP and SAP in DzO-based solutions compared to the amide I1 band of FP in HzO-based solution is expected. It hasbeen well documented that theabsorbance maximum frequency of the infrared amide I band is determined by the predominant secondary structure in the proteins (27, 29-31, 38). For proteins in HzO-based solutions, the amide I band maxima of proteins with predominant a-helix structure are usually found a t 1656 2 2 cm-’ and those with predominant p-sheet structure are usually found between 1643 and 1631 cm-l(24,29).For proteins inD2O-based solutions, the amideI‘

6426

Effects of Ca2+,M e , and PC on Pentraxins I

I

I

I

"- CRPICa++lPC

I

I

I

I

I

-CRPICa++

TABLE I Assignments for deconvoluted infrared amide I band components of human C-reactiue protein and serum amyloid P component in D20-based solutions Serum amyloid P component

C-reactive protein Frequency" cm"

Assignment

Frequency"

Assignment

cm"

1625(6*,6) /%Strand /"rand 1632(3*,3) p-Strand 1645 Unordered Unordered a-Helix 1652(l*,1) a-Helix Type I11 turn 1660 Type 111 turn or 310-helix or 3,,-helix 1669 (70*, 71)P-Turn 1668 (73*, 73)p-Turn 1678 (7*, 7) p - k 1678 (81*,81) p-Turn 1688 p-Turn P - k 1687 1695 p-Strand 1695 p-Strand a The values in parentheses are from proteins with Ca2+ (marked with asterisk) and Ca2+plus PC. 1631(2*,2) 1646 1655(4*,4) (1660*, 2)

I 1700 1600

CRP

I

1650 WAVENUMBER (cm-1)

FIG.2. The original, Fourier self-deconvoluted, and inverted

second-derivative amide I infrared spectraof human CRP with

PC.

or without Cas+and CRP (- - - - -); CRP with 2 m Ca2+(-); CRP with 2 m Ca2+ plus 1 m PC (- - -). Zbp, the original spectra;

middle, Fourier self-deconvoluted spectra; bottom, inverted second-derivative spectra. The reproducibility of all spectra was confirmed by at least two independent measurements. band maxima of proteins with predominant a-helix structure are found between 1654 and 1648 cm-l and those with predominant @sheet structure are found between 1640 and 1627 cm-l (27,32). The amide I' band maxima of CRP and SAP at 1633 m-' indicated that thep-sheet structure is the predominant secondary structure in theseproteins. Fourier Self-Deconvolution and Second-derivative Annlysis-To resolve the overlapping band components under the amide I contour, two mathematicalband-narrowing procedures, namely Fourier self-deconvolution and second-derivative, have been extensivelyused inprotein infrared studies(27, 28, 33). Fig. 2 shows a n overlay of the original, Fourier selfdeconvoluted, and second-derivative amide I' spectra of CRP with or without Ca2+ PC. andThe second-derivative spectra are presented as the inverted form for easier comparison. Both Fourier self-deconvolution and second-derivative analysis revealed the amide I' band components ascribed to the p-sheet structure (1695 and 1631 cm"), a-helix (1654 cm-l), p-turn (1688, 1678, and 1670 cm-l), and unordered structure (1646 cm-') (24, 27, 33, 35). A small band at 1660 cm-l was also evident when Ca2+was present. The precise assignment for the 1660 cm-' band cannot be made for the following reasons. Results of our earlier studies(24, 29) and studies of other investigators (39-42)have shown that amide I band components at 1663 2 3 cm-1 are associated with three different types of secondary structures: the 310-helix structure as in a-lactalbumin and deoxyribonuclease I3 and in peptides containing a-aminoisobutyric acid (42);the aII-helix structure as in bacteriorhodopsin (39-41); and type I11 turn as in cytochrome c (35). The bands arisingfrom these structurescannot be distinguished on the basisof their frequency. Therefore, we tentatively assigned the 1660 cm" band to type I11 turn as the most likely structure. The bands between 1620 and 1600 cm-l have been generally assigned to side chain vibrationsof amino acid residues (27,32, 34, 43). Although exclusive correspondence between the high A. Dong, P. Huang, B. Caughey, and W. S. Caughey, unpublished

data.

1700

1650

It

WAVENUMBER (cm-1)

FIG.3.The curve-fittingof inverted second-derivative spectra of human CRP with or without Caa+ and PC. The curve-fitting procedure was carried out as described under "Materials and Methods," and the deconvoluted band frequenciesare listed in Table I.

wave number p-structure band component and antiparallel p-sheet has not been solidly confirmed experimentally (44), theoretical analyses suggest it is most likely associated with the antiparallel p-sheet structure (31, 45). Suggested assignments for the deconvoluted amide I' band components are summarized in Table I. Quantitative analysis (Fig. 3) was carried out with the second-derivative spectrum by use of Gaussian curve-fitting procedures.Bands below 1620 cm", which are not amide I components, were includedin thecurve-fitting to avoid possible distortion. These results support the conclusion that human CRP in solution is also composed predominantly of p-sheet structure.Turn,a-helix,and unordered structures were observed in lesser amounts (Table 11).A similar, but somewhat less reproducible, secondary structure composition was self-deconvolutiodcurve-fitting also obtainedusing the Fourier method of Susi and Byler (27) (data not shown). Fig. 4 presents anoverlay of the original, Fourier self-deconvoluted, and second-derivative spectra of SAP with or without Ca2+ and PC in the amide I' region. Bothband-narrowing analyses revealed the amide I' band components ascribed to the P-sheet structure (1695, 1632, and 1625 cm-'1, a-helix (1652 cm-l), p-turn (1687,1678,and 1668 cm"), and unordered structure (1645 cm") (24, 27, 33). The band at 1660 cm-', which we tentatively assigned to type I11 turn, was also evident. Fig. 5 shows curve-fitted invertedsecond-derivative spectra. The estimated secondary structure composition of human

6427

Effects of ea2+,M e ,and PC on Pentraxins TABLEI1

Pentraxin

I

SAP

Secondary structures of pentraxins C-reactiveprotein and serum amyloid P component in solutions as estimated by amide I infrared second-derivativeanalysis The values represent the percentage of the total integrated intensity of the second-derivative amide I spectral bands which corresponds to bands assigned to the designated secondary structure. Secondary structure

Turn

P-Sheet a-Helix

Unordered

%

13.7 Human CRP23.3 50.0 13.0 CRP/Ca2+ 27.6 45.4 9.2 17.1 27.3 45.3 10.3 CRP/Ca2+/PC Human SAP 24.955.910.1 8.9 26.0 52.3 12.8 SAP/Ca2+ SAP/Ca2+/PC 8.9 26.0 52.9 12.8 Hamster 11.6 FP" 28.4 50.8 9.2 FP/Ca2+ 50.0 10.8 9.4 10.8 28.9 50.9 FP/Ca2+PC a

17.8

SAP/Ca++/PC

9.1

29.8

'

9.4

)O

1650

1 00

WAVENUMBER (cm-1)

Data from Dong et al. (24).

FIG.5.The curve-fittingof inverted second-derivative spectra of human SAP with or without Ca2+ and PC. The curve-fitting procedure was camed out as described under "Materials and Methods," and the deconvoluted band frequencies are listed in Table I.

-SAP/Ca++

CRP N

d

w

m

5

2 0

>

m p:

m 4

-Ca++-depleted with Mg++ " "

I 1650

1700

1600

1650

WAVENUMBER (cm-1)

FIG.4.The original, Fourier self-deconvoluted, and inverted second-derivative amideI infrared spectra of human S A P with or without Ca2+ and PC.SAP (- - - - -); S A P with 2 m~ CaZ+(-1; S A P with 2 m~ Ca2+plus 1 m~ PC (- - -). Top, the original spectra; middle, Fourier self-deconvoluted spectra; bottom, inverted second-derivative spectra. The reproducibility of all spectra was confirmedby at least two independent measurements.

S A P was similar to those estimated for human CRP and hamster FP (Table 11) and is consistent with a predominantly antiparallel p-sheet structure insolution as well as in crystal(25). A similar secondary structure composition was also estimated using the Fourier self-deconvolutiodcurve-fittingmethod of Susi and Byler (27) (data notshown). Calcium-dependentAmide Z Spectral Changes-Calcium plays an importantrole in thebiological function of pentraxins (8, 9, 11, 14, 15).By comparison of amide I spectra of CRP and S A P with or without Ca2+, significant spectral differences were consistently seenin regions assignedto p-sheet,a-helix, p-turn, and unordered structures (Figs. 2 and 4). The Ca2+dependent spectral changes were seen clearly even without resolution enhancement. The most remarkable changes were detected at regions assigned to p-sheet and unordered structures. Upon Ca2+binding, the strong bandsascribed to p-sheet are blue-shifted about 1cm-' accompanied by narrowing, while the bands arisingfrom a-helix are red-shifted about 1 cm" in both CRP and SAP (Table I). Furthermore, a new band a t 1660

I

I

I

I

I

I

I

I

1700

WAVENUMBER (cm-1)

FIG.6. Comparisons of the second-derivative spectraof Cas+depleted human CRP and SAP in the presence or absence of 2 m~ Mg2+.

cm-l appeared in the spectra of CRP, whereas a significant intensity increase a t 1660 cm" band was observed in SAP. These results provided strong evidence for conformational changes due to Ca2+binding in both proteins. Phosphorylcholine-dependent Amide

Z Spectral

Changes-

Relatively small but fully reproducible spectral changes in the amide I region were observed in CRP following the addition of PC in thepresence of Ca2+.The PC-dependent amide I spectral changes are concentrated between 1673 and 1640 cm", the regions assigned to turn, a-helix, and unordered structures. The curve-fitting analysis revealed that the changes resulting from PC binding are mainly the frequency shift and relative intensity changes of two bands ascribed to p-turn structure near 1663 and 1671 cm-l (Table I). Such frequency and intensity changes in P-turn-related bands usuallysuggest relative movements in main chain structures (29); in the case of CRP, the antiparallel P-sheet structure. A PC-dependent change is not apparent in the spectraof SAP. Magnesium-inducedAmide Z Spectral Changes-Fig. 6 presents anoverlay of the second-derivative amideI' spectra of CRP and S A P with orwithout M2+.Significant spectral changes involving regions assigned to the p-sheet and a-helix

Effects of Ca2+,M e ,and PC on Pentraxins

6428

1 CRPID20

.

.

N

#

V

11111 111111 1650 1600

1700

WAVENUMBER (cm-1)

FIG.7. Comparisons of the second-derivative spectra of Ca2+depleted humanCRP and SAP in D,O-based solutions and hamster Fp in H,O-based solution. The spectrum of hamster FP is from Dong et al. (24). a,a-helix; U , unordered structure.

structures were observed in CRP, whereas a complete superimposition between the spectraobtained with or without M e was observed in SAP. The spectralchanges induced by M e in CRP differ markedly from that induced by Ca2+,especially at the p-structureregion. While binding of Ca2+shifted the strong p-structure-related banda t 1631 cm-' toward the blue, binding of M e shifted the band slightly to the red. This finding suggests that althoughsignificant conformational changes can be or M e , the role played by Ca2+ induced by binding either Ca2+ may not be duplicated by M 2 + . DISCUSSION

Comparison of Secondary Structures of Human CRR Human S M , and Hamster FP-It has been considered likely that the pentraxins CRP, SAP, and FP have similar protein structures on the basisof their high sequence homology (5-7) and similar pentameric structures (1, 2). A detailed comparative study of the secondary structures of pentraxins has not been conducted. However, using far-UV CD spectroscopy, Young and Williams (23) estimated similar p-sheet and a-helix structures for human CRP and SAP. However, using second-derivative Fourier transform-IR spectroscopy Dong et al. (24)estimated that hamster FP contains much lower amounts of a-helix than suggested byYoung and Williams (23) for CRP and SAP (11% for FP compared with 34% for CRP and SAP). The present study shows that humanCRP and SAP and hamsterFP have similar amide I spectral patterns(Fig. 7). The spectrumof each protein exhibits a very strong low wave number p-structurecomponent near 1634 cm-l and a weak high wave number p-structure component near 1695 cm-l as well as a weak band arisingfrom a-helix and a weak band arising from unordered structure. Quantitative analysis(Table 11)revealed that humanCRP and S A P and hamster FP are all predominantly composed of antiparallel p-sheet structure (5045%) with smallamounts of a-helix (9-13%) and unordered (9-18%) structures. These results support theidea that pentraxins have similarsecondary structure folding patterns. Nevertheless, there arenotable difS A P and hamster ferences amongthe three pentraxins. Human FP both exhibit a p-structure-related band near 1625 cm-l, which has been attributed to the"exposed" or strongly hydro-

gen-bonded p-sheet structure (46, 47). No similar band near 1625 cm-' was found in the spectrum of human CRP. Under identical conditions, the major p-structure component of S A P exhibits a slightly higherfrequency (= 1 cm-') than thatof CRP (Table I). Furthermore, human CRP contains a considerably higher percentage of unordered structure than either human SAP or hamster FP (Table 11). We should mention here that the 34% a-helix structure estimated for human CRP and SAP in solution by Young and Williams (23) using far-UV CD spectroscopy is apparently an overestimation on the basisof the recentx-ray crystallographic analysis of human SAP (25). This overestimation of a-helical content by far-UV CD may have resulted from the aromatic side chain clusters formed in these proteins (24, 48, 49). The amino acid composition of human CRP derived from the complementary DNA sequence shows that it contains 8 Tyr, 13 Phe, and 6 Trp (5, 50). The location of many of these residues sequentially close to each other suggests they may form aromatic side chain clusters. Several methods have been developed in the pastfew years on the basis of empirical studies to estimate the relativecontributions of different types of secondary structures inproteins from their infraredamide Ispectra (32,33,51-53). These methods can be classified into two categories. First are the deconvolutional methods includingFourier self-deconvolutiodcurvefitting (27,321 and second-derivative analysis (33). Second are the semi-deconvolutional methods including the partial leastsquares analysis (51), factor analysis (52), and data base analysis (53). The advantages and disadvantagesof these methods have been discussed in recentreview articles (54,55).However, due to theempirical nature and uncertainty in data processing and interpretation associated with each of these methods, the evaluation should ultimately be based on how successful these methods are in estimating the secondary structure of a protein prior to any high resolution information becoming available. The excellent agreement between the secondary structure of pentraxin hamster FP estimated on the basis of IR-secondderivative analysis (24) and recentx-ray crystallographic analysis of human S A P (25) provides further supportfor the useof the refined IR-second-derivative anaysis (24, 35) as a reliable technique for estimating protein secondary structure despite its inherent shortcomings, namely thepotential interference of weak positive side lobes associated with the second derivative. IR-second-derivative analysis is particularly useful for the detection of subtle changes in structure such as the effects of Ca2+,M$+, and PC observed in this study. Similar secondary structure compositions for CRP and SAP were also obtained using the Fourier self-deconvolutiodcurve-fittingmethod of Susi and Byler (27) (data not shown). Effects of Hydrogen-Deuterium Exchange on Amide I Band Frequencies-Unlike manyother proteins studiedin DzObased solutions (27,34, 56),the frequencies for the majority of deconvoluted components of CRP and SAP appear not to be significantly affected by hydrogen-deuterium exchange except for the bandsascribed to the a-helix and unordered structures (Fig. 5) when compared with the spectrum of hamster FP in HzO-based solution (24). The bandfrequencies assigned to different types of secondary structures in human CRP and S A P deviate somewhat from values reported for deuterated proteins (27, 32). Especially noteworthy is our assignment of the high wave number p-structure component to the band near 1695 cm-l instead of the band at 1675 2 4 cm-l (27, 32). This assignment is supported by the infrared studyof concanavalin A (46), a protein with similar secondary structure to SAP (25). Arrono and co-workers (46) have shown that the deconvoluted amide I spectrum of concanavalin A exhibits a band ascribed to the high wave number p-structure component at 1694 cm-' in

Effects of

PCM$+, Pentraxins and on ea2+,

TABLEI11

6429

in H20-basedsolution. functional importance of Due to the apparent structural and Ca2+binding in pentraxins, many attempts have been made to Pentraxin Calcium Magnesium Phosphorylcholine localize the possible binding site(s) by searching for the amino acid sequence resemblance between pentraxins and therecogHuman CRP +++ ++ ++ +++ nized consensus pattern in EF-handed (relatedto the E and F Human SAP f +++ Hamster FPa +++ helices of parvalbumin) Ca2+ binding domainsof parvalbumin and calmodulin (3, 4, 24, 60, 61). The recent x-ray crystalloData from Dong et al. (24). graphic analysis (25) revealed that the Ca2+ binding site of both H 2 0 and D20 solutions. They attributed the lack of iso- human SAP consists of two loops from distant peptide segtopic effects on frequencies of p-structure-related bands tolow ments andcoordinates two calcium ions in a manner similarto hydrogen-deuterium exchange rate of p-sheet structure. As- the Ca2+ binding sites of concanavalin A (62,631 and pea lectin suming that thefrequencies of the deconvoluted band compo- (64). Thus the Ca2+-binding site of human SAP (and probably nent for the a-helix structureof human CRP and S A P in H20- pentraxins in general) differs markedly from the recognized based solutions are similar to the corresponding band consensus pattern of amino acid residues in the EF-handed frequency in hamster FP, which is most likely true judged on Ca2+-bindingproteins (65, 66). the basis of the large percentage of amino acid sequence homology (7) and the similarity insecondary structure composiREFERENCES tions (Table 11),the a-helix structureof SAP seems to be more 1. Osmand, A. P., Friedenson, B., Gewurz, H., Painter, R. H., Hofmann, T., and affected by hydrogen-deuterium exchange than that of CRP Shelton, E. (1977)Pm. Natl. Acad. Sci. U. S. A. 74,739-743 2. Coe, J. E.,Margossian,S . S., Slayter, H. S.,and S o g n , J. A. (1981) J. Exp. Med. (Fig. 7). In comparison with hamster FP, a red shift of 5 cm-l 163,977-991 (from 1657 to 1652 cm-') in bandfrequency assigned to a-helix 3. Nguyen, N.Y., Suzuki, A,, Cheng, S.-M.,Zon, G., and Liu, T.-Y.(1986) J . Biol. Chem. 261, 10450-10455 was observed in human SAP, whereas only about 2 cm-l red 4. Nguyen, N. Y., Suzuki, A., Boykins, R. A., and Liu, T.-Y.(1986) J. Biol. Chem. shift (from 1657 to 1655 cm-l) was observed in human CRP 261, 10456-10465 after 24-h hydrogen-deuterium exchange. This difference may 5. Woo, P., Korenberg, J. R., and Whitehead, A. S. (1985) J. Biol. Chem. 260, 13384-13388 be because the a-helical structure of S A P is more exposed to 6. Whitehead, A. S . , Zahedi, K., Rita,M.,Mortensen, R. F.,and Lelias, J. M. solvent than the a-helical structure of CRP. (1990) Biochem. J. 266,283-290 Ca2+- and PC-dependent Conformational Changes-Ca2+ is 7. Dowton, S. B., and Holden, S . N. (1991)Biochemistry 30,9531-9538 8. Gotschlich,E. C., and Edelman, G. M. (1967)Proc. Natl. Acad.Sci. U. S. A. 67, essential for a variety of binding activities of CRP (8, 12-15, 706-712 57), SAP (9, 58),and FP (2). It has been suggested that Ca2+ 9. Painter, R. H., De Escallon, I., Massey, A,, Ointeric, L., and Stern, S. B. (1982) Ann. N. Y. Acad. Sci. 389, 199-215 acts as a n allosteric effector which induces the proper conforCoe, J. E. (1983) Contemp. Top. Mol. Immunol. 9, 211-238 mation of the PC-binding site (57). Ca2+-dependent conforma- 10. 11. Coe, J. E., and Rose, M. J. (1983) J. Exp. Med. 167, 1421-1433 tional changes havebeen observed in human CRP (23,59) and 12. Robey, F. A., Jones, K. D., Tanaka, T., and Liu, T.-Y.(1984)J. Biol. Chem. 269, 7311-7316 hamster FP (24). Present findings provide evidence of Ca2+Du Clos, T.W. (1989) J. Immunol. 143, 2553-2559 dependent conformational changes in human CRP and SAP. 13. 14. Du Clos, T. W., Zlock, L. T., and Rubin, R. L. (1988)J . Immunol. 141, 4266Exposure of CRP andSAP to Ca2+ caused changes in the amide 4270 15. 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Res. 14, 44-59 copy are consistent with the Ca2+-dependent binding activities only CRP 21. Coe, J. E., and Rose, M. J. (1985) J. Clin. Inuest. 76, 66-74 of these proteins(8-15,18). Among the three proteins 22. Coe, J. E., and Rose, M.J. (1990) J. Exp. Med. 171, 1257-1267 binds M$+ as well as Ca2+. However, the amide I spectral 23. Young, N. M., and Williams, R. E. (1978) J. Immunol. 121, 1893-1898 changes induced by binding of Mg2+ differ markedly from those 24. Dong, A., Caughey, B., Caughey, W. S., Bhat, K. S., and Coe, J. E. (1992) Biochemistry 31,9364-9370 induced by binding of Ca2+. Therefore, it is unlikely that the 25. Pepys, M. B. (1993) in Abstracts of 7th InternationalSymposium on Amyloifunctional role played by Ca2+can also be carried outby M$+. dosis, Queen's University, Kingston, Ontario, July 12-15 26. Parker, F. S. (1983) Applications of Infrared, Ramanand Resonance Raman Effects of PC on the conformation-sensitive amide I region difSpectroscopy in Biochemistry,Plenum Press, New York fer among the three proteins. Large PC-dependent conforma- 27. Susi, H., and Byler, D. M. (1986) Methods Enzymol. 130, 290-311 tional changes were observed in hamster FP and human CRP, 28. Surewiu, W. K., and Mantsch, H. H. (1988) Biochim.Biophys.Acta 962, 115-130 whereas almost no PC-related conformational changes were 29. Dong, A., and Caughey, W. S . (1994) Methods Enzymol. 232, 139-175 detected in humanSAP.The lack of a PC-dependent conforma- 30. Miyazawa, T., and Blout, E. R. (1961)J . Am. Chem. Soc. 83,712-719 tional change in human SAP is consistent with no report of a 31. Krimm, S., and Bandekar, J. (1986)Adu. Protein Chem. 38, 181-364 32. Byler, D.M., and Susi, H. (1986) Biopolymers 26, 4 6 W 8 7 PC-related activity of SAP in the literature. 33. Dong, A., Huang, P., and Caughey, W. S . 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(1982)Science 216,407-408 cause of the difference in PC-dependent conformational 41. Hans, P. I., and Chapman, D. (1988) Biochim. Biophys. Acta 943,375-380 changes between human CRP and hamster FP. Unfortunately, 42. Kennedy, D. F., Crisma, M., lbniolo, C., and Chapman, D. (1991) Biochemistry 30,6541-6548 the aggregation of CRP and SAP at high concentration (>I5 43. Chirgadze, Y. N., Fedorov, 0. V., and Trushina, N. P. (1975) Biopolymers 14, mg/ml), especially in thepresence of Ca2+, prevents their study 679-694 Amide I inbared spectral changes of pentraxins induced by calcium, magnesium, and phosphorylcholine

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