Apr 15, 2015 - Vel Ala Ala ASP Lys HIS Tyr Gln CLu Cln Ala Lya. 10.9 13.7 13.9 10 8 NQ. 6 3 8.3 2.9 7.5 6.6 6 8 NQ. Lys HI. Cln Clu Tyr Lys dln Clu Oln Glu ...
Vol. 263, No. 11, Issue of April 15,pp.Printed 5075-5082, 1988 in U.S.A.
THEJOURNALOF BIOLOGICAL CHEMISTRY
Complete AminoAcid Sequence of Streptococcal PepM49 Protein, A Nephritis-associated Serotype CONSERVED CONFORMATIONAL DESIGN AMONG SEQUENTIALLY DISTINCTMPROTEINSEROTYPES* (Received for publication, August 31, 1987)
Kiran M. KhandkeS, Thomas Fairwells, A. Seetharama Acharyallll ,Benes L. Trus**, and Belur N. ManjulaS From $The Rockefeller University, New York,New York 10021, the $Molecular Diseases Branch, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892, the lIDiuision of Hematology, Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461, and the **Computer Systems Laboratory, Division of Computer Research and Technology, National Institutes of Health, Bethesda, Maryland 20892
The complete amino acid sequence of PepM49, a (1-6). It is very well recognized that while a large number of peptic fragment of the group A streptococcal type 49 streptococcal serotypes are associated with rheumatic fever, M protein, the antiphagocytic cell surface molecule of only a limited number are associated with infections of the the bacteria, is described. This fragment retains the skin and theircomplications such as acute glomerulonephritis opsonic antibody epitope of the native molecule. The (3-8). The streptococcus evades the host's immune system by sequence of PepM49, as determined by automated Ed- the expression of an immunologically diverse surface antigen, man degradations of the uncleaved molecule, and its the M protein.' M protein functions as a major virulence tryptic and chymotryptic peptides, consists of a total factor for these organisms by virtue of its ability to impede of 143 residues (M. = 17,187). PepM49, a nephritis- their ingestion by human phagocytes (9). Over 80 serological associated M protein serotype, exhibits significant in- variants of the Mprotein have been recognized to date. ternal homology in its sequence. However, identical Although immunological cross-reactions occur among hetersequence repeats of the kind seen in the rheumatic ologous M protein serotypes (9-15), the immunity conferred fever-associated serotypes M5, M6, and M24, are abby the induced antibodies to a given M type is essentially sent in PepM49. PepM49 exhibits varying degrees of type-specific (9). homology with the M5, M6, and M24 proteins, which Structural andphysicochemical studies over the past decade is consistent with the existence of variable and conserved regions in the M protein molecule. Predictive concerning mainly three of the rheumatic fever-associated analysis as well as CD measurements revealed a high serotypes, 5, 6, and 24 (15-22)have revealed that the M propensity of the PepM49 molecule to assume an a- protein of the group A streptococcus is a dimeric a-helical helical conformation. Furthermore, a heptad periodic- coiled-coil moleculeand exists as aflexible fibrillar structure, ity of the nonpolar residues, a characteristic of a- with its NH,-terminal region being distal to thebacterial cell helical coiled-coil proteins,extendsoverthe entire surface (16, 20, 23). Immunological and DNA hybridization length of the PepM49 protein. The differences in the studies have revealed that regions within the COOH-terminal nonpolar residue distribution divide the PepM49 se- half of the M molecule are conserved among many M seroquence into three distinct domains, similar to those types and those inthe NH,-terminal region are more variable, seen earlier in the M5 and M6 proteins. Together, these the NH2-terminal 25% being hypervariable (13, 15, 24, 25). studies establisha conserved conformational design for The NHz-terminal half of the M protein also contains the the sequentially diverse M protein serotypes. However, type specific opsonic antibody epitope of the native molecule the pattern of heptad periodicity in the PepM49 pro- (26-28) and removal of this region of the molecule bypepsintein is quite distinct from that present in the PepM5 treatment of the streptococci results in the loss of antiphagand M 6 proteins, suggesting distinct differences in ocytic activity (20,29). Thus, theNH,-terminal half of the M structural featuresamong conformationally similar M protein appears to play a major role in the functional aspects protein serotypes. This may have relevance to the pathof the molecule, namely, antigenic variation, the presence of ological differences associated with these M protein opsonic antibody epitope, and antiphagocytic behavior. serotypes. In contrast to the rheumatic fever-associated M protein serotypes, structural information on the nephritis-associated M proteinserotypes is limited to theNHz-terminal sequences of the pepsin-derived fragments (PepM molecules) of two Acute rheumatic fever and acute glomerulonephritis are serotypes, M1 (30) and M49 (31). Thepattern of heptad two of the major sequelae of group A streptococcal infections periodicity in these NH,-terminalsequences (residues 1-55 of * This work wassupported by United States Public Health Service Grants HL-36025 (to B. N. M.) and DK-35869 (to A. S. A.). The Aviv 60 DS CD spectropolarimeter was purchased with funds from National Science Foundation Grant PCM-8400268. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 11 Established Fellow of the New York Heart Association.
' The abbreviations and trivial names used are: M6, the molecule representing the complete M6 protein;PepM protein, M protein isolated from streptococci by limited proteolysis with pepsin; DHP, dihydroxypropyl; RPHPLC, reverse-phase high performance liquid chromatography; TFA, trifluoroacetic acid; TPCK, L-l-tosylamido2-phenylethyl chloromethyl ketone; TLCK, N"-p-tosyl-L-lysinechloromethyl ketone; PTH, phenylthiohydantoin; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
5075
5076
Conserved Conformational Designof Streptococcal M Proteins 20
10 Val Glu Lya
LysVal Glu Ala Ala Glu Ann Asn Val Ser Ser Val Ala Arg
""""""""""""-
ArgGlu Lys Glu Leu Tyr Asp Gln
I
I
I
DHP-TS 30 40 60 [ l e Ala Asp Leu I h r Asp Lys Asn Gly Glu Tyr Leu Glu Arg I l e Gly Glu Leu Glu Glu Arg Gin Lys Asn Leu
"""""""_"""""
FIG. 1. Summary of the evidence for the complete sequence of streptococcal PepM49protein. Peptide designations are immediately under the sequence and according to the enzyme used for cleavage. DHP-T,tryptic peptides of dihydroxypropylated PepM49; C, chymotryptic peptides. Dashed line repDHP-T1 resents the sequence determinedby Edman degradation of the uncleaved PepM49 protein. All peptides, except C3, were sequencedthrough their COOHterminal residue (indicated by solid line).
..
.. DHP-TB
DHP-TI1
eo
DHP-TI0
70
Glu Lys Leu Glu His Gln Ser Gln Val Ala Ala Asp Lys H i s Tyr Gln G l u Gln Ala Lys
LysH i s Gln Glu Tyr
" " " " "
DHP-T10
so
90
Lys Gln Glu Gln G l u Glu Arg Gln Lys Asn Gln Glu Gln Leu Glu Arg Lys Tyr G l n Arg Glu Val Glu Lys Arg
"
I
DHP-TlO DHP-TZ
I
DHP-TL c1 110
Tyr
Gln Glu G l n Leu Gln Lys Gln Gln Gln Leu Glu
'Ihr
Glu Lys Gln I l e
Ser Glu Ala Ser Arg 4 s Ser Leu
DHP-TO
..
DHP-TI
130 140 Ser Arg Asp Leu G l u Ala Ser Arg Glu Ala Lys Lys LysVal Glu Ala Asp Leu I I DHP-TI DHP-T7
c2
t
PepM5(17), M6 (19), andthepartial sequences of the PepM24 protein (21, 22) to understand the structural basis for the immunological diversity of these M proteinmolecules. MATERIALS AND METHODS
RESULTS~
Amino Acid Sequence of PepM49"The complete amino acid sequence of PepM49 together with the dataleading to its determination is shown in Fig. 1. PepM49 contains 11 arginines, 19 lysines, 48 glutamates (Glu + Gln), and no methionine and tryptophan, thuslimiting the choice for obtaining a small numberof peptides to cleavage at arginyl-peptide bonds. In our earlier studies on the sequence analysis of streptococcal PepM5 (17) and PepM6 proteins (15), blocking their amino groups by reductive dihydroxypropylation (32) proved to be a very valuable procedure to limit the tryptic cleavage to their 50 IO0 arginyl residues. We have now employed the same strategy to PepM49 obtain thearginine peptidesof PepM49. The sequences of the FIG. 2. Internal homology in PepM49, as revealed by dot arginine peptides of dihydroxypropylated PepM49, together matrix analysis (35).The sequence of the PepM49moleculeis presented on both the horizontaland the vertical axis. The numbers with the previous knowledge of the NH2-terminal sequence of on the axes referto residue numbers within the sequence. Scores are the uncleaved protein (31), provided the framework for the accumulated from a sequential comparison of segments of 20 residues, sequence presented in Fig. 1. Overlapping peptides were obusing mutation data matrix (53). Scores greater than or equal to the tained by limited proteolysis of unmodified PepM49 protein mutation data matrix score of 25 are displayed by dots in a matrix with chymotrypsin. The results thus obtainedestablish plot, the position of the dots corresponding to the middle of each segment in any given pair. The continuous diagonal line represents PepM49 to be a 143-residue protein. Molecular Size of the PepM49 Molecule-The molecular the line of identity as a result of comparison of the sequence with itself. Internal homology withinthe sequence is revealed by the series mass of the PepM49 protein calculated from its amino acid of diagonals offset from the major diagonal. sequence is 17,187 daltons. This value is significantly smaller than the20 kDa estimatedfrom the SDS-PAGE analysis(31). PepMl and residues 1-60 of PepM49) was found to be more Similar over-estimatesof molecular mass by SDS-PAGE have similar to each other than to those in the PepM5, PepM6, been seen for other M proteins (19), as well as other coiledand PepM24 proteins (31), suggesting distinct differences in coil proteins (33, 34), and may be a reflection of the fibrous the structural features of the nephritis and rheumatic fever- nature of their structure. Sequence Repeats in PepM49-A dot matrix analysis (35) associated M protein serotypes. In this report we present the complete amino acid sequence of PepM49, a peptic fragment of the PepM49 sequence revealed significant internal homolof the type 49 M protein that retains the opsonic antibody ogies which are localized in essentially two distinct regions epitope of the native molecule. PepM49 is thus the firstsuch * Portions of this paper (including "Experimental Procedures," part biologically important fragment of a nephritis-associated M of "Results and Discussion,"Figs. 1 and 2, andTables 1-4) are protein serotype to be sequenced completely. This sequence presented in miniprint at the end of this paper. Miniprint is easily has now been examined to determine the extentto which the read withthe aid of a standard magnifying glass. Full size photocopies heptad periodicity extends within the PepM49 molecule. In are included in the microfilm editionof the Journal that is available addition, PepM49 has been compared with the sequences of from Waverly Press.
Conserved Conformational Design
of Streptococcal M Proteins
within the molecule (Fig. 2). The homologies in the NH2terminal region (region A) are limited. However, those in the region COOH-terminal to this (region B) are more extensive and more numerous. Region B contains an inexact repeat represented by segments 41-76 and 77-112 (50% identity, which includes the hexapeptide EERQKN) (Fig. 3A). Furthermore, segment 25-60, which overlaps with segment 4176, exhibits 33% identity with segment 77-112 and segment 61-76 is homologous with segment 127-142. Also, different sections of segment 127-143 are repeated elsewhere within the molecule (Fig. 3B). Sequence Homology of the PepM49 Protein with Other M Proteins-To determine the homology with the otherM proteins, the PepM49 sequence was compared to the sequences of PepM5 (17), M6 (19), and thepartial sequence of PepM24 (22) by the Fastp program (36). These analyses revealed that PepM49 shows the least homology with PepM5; 19% identity was observed between segment 1-103 of PepM49 and 97-196 of PepM5 (Fig. a), whereas segment 107-132 of PepM49 showed 38% identity with peptide CB3 of the M24 protein (26-residue overlap) (Fig. 4B), and the complete sequence of
,,
A. 127-142
~
~
'1
u
R
Q
E
a
d
A
D
41-70
G E L E E R Q K N L E K L E H Q S Q V A A D K ~ Y Q E Q A K K ~ Q E Y K
77.112
Q E Q E E R Q K N Q E Q L E R K Y Q R E V E K R Y Q E Q L Q K Q Q Q I . E
16-60
Q I A D L T D K N G E Y L E R I G E L E E R Q K N L E K L E H Q S Q V A
5077
PepM49 exhibited 33% identity with segment 156-304 of M6 (149-residue overlap) (Fig. 4C).More significant are the identities in the last 30 residues within this stretch. 73% identity is present between segment 114-143 of PepM49 and 275-304 of the M6 molecule. The results of the Fastp program display only the region having maximal homology.M6 protein has three sequence repeat regions A, B, and C (19). Segment 275-304 of the M6 protein, which is within the C-repeat region of the molecule, is essentially repeated once more at residues 233-262. Hence, segment 114-143 of PepM49 is homologous with two segments of the M6 molecule (73% identity with segment 275-304 and 70% identity with segment 233-262, the sharedidentities being 60%) (Fig. 4D). Thus, PepM49 may be considered to contain 1 equivalent of the M6 C-repeat region. Secondary Structure of the PepM49 Protein-Predictive analysis of the PepM49 sequence by the method of Garnier et al. (37) revealed a very high a-helical potential virtually over its entire length (Fig. 5). This result is further supported by the results of the circular dichroism spectra (Fig. 6). PepM49 in 10 mM phosphate buffer, pH 7.0 (curve a), was
VEKKVEAAENNVSSVARREKELYDQIADLTDKNGEY HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHCCCCHH
ao
LERIGELEERQKNLEKLEHQSQVAADKHYQEQAKKH HHHHHHHHHHHHHHHHHHHHHHHHHBHHHHHHBBHH
7a
B. I K K V F A ~
3-7 27-29 69-71 119.121
E A
s n
FIG. 3. Sequence repeats in PepM49. A , homology between segments 41-76 and 77-112 of PepM49. Identical residues are boxed. 18/36 residues (50%) are identical. Other parts of the molecule showing homology with these segments are also shown in this figure. B , regions of the PepM49 protein bearing identity with different parts of segment 127-143 are shown. 3 0 A.
PepM49 PepM6
QEYKQEQEERQKNQEQLERKYQREVEKRYQEQLQKQ HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH
108
QQLETEKQISEASRKSLSRDLEASREAKKKVEADL HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH
14a
FIG. 5. Secondary structural characteristicsof the PepM49 protein as determinedby the predictive method of Garnier et al.(37). Amino acid residues are indicated by single letter code. The characteristic assigned to each residue indicates the potential of that residue. The assignments H a n d C represent a-helicaland coil potential, respectively. IO
2 0
.........
. . . . . .:
:
80
..
BO
60
.
:
. . . .
. . : . : . v :
140
130
PepM49
ADKHYQEQAKKHQEYKQEQEERQKNQEQLERKYQREVEKRYQEQ KIAKEQENKETICTLKKILDETVKDKLAKEQKSKQNIGALKQEL
. .. .. . . . . : .
LOO
..
. .. . . . . . : . : . . . .
170
180
110
150
100
90
PepMK
1 BO
190
120
130
EKRYQEQLQKQQQLETEKQISEASRKSLSRDLEASR
PepM49
:
. x:.::.
: : . . .:...:x..
NFSTADSAKIKTLEAEKAALEAEKADLEKALEGAM
PepM24/CB3
20
10
30
20
10
C .
:
120
110 70
8.
_ v . : . . . .
LEREVQNTQYNNETLKIKNGDL---TKELNKTRQELANKQQESKENEKALNELLEKTVKD 100
FIG. 4. Comparison of the amino acid sequence of PepM49 with (A) PepM5 (17), (B)PepM24 (22), and (0 M6 (19). by the Fastp program (36).Only the portions of the two sequences containing homology are shown. D,the two segments of the M6 protein bearing extensive identity with the PepM49/114-143 are shown. The identities between the PepM49 sequence and the two segments of the M6 protein are boxed.
4 0
VEKKVEMENNVSSVARREKELYDQlADI.TDKNGEYLERIGELEER~LEKLEHQSQVA
4 0
30
6 0
I'epM49
VEKKVEAAENNVSSVARREKEI,YDQIADLTDKNGEYLERICEL----EERQKNLEKLEHQ
MB
VKDKIAKEQESKETIGTI,KKTLDETVKDKIAKEQESKETIGTLKKILDETVKDKIAREQK
. . .. . . . . . . . . . . . . . . . . . . . .. . .. .. . .. . .. . .. . 1BO
1 7 0
I 8 0
I 9 0
BO
70
80
90
. .. .. .
200 100
...
210
I 1 0
PepM49
SQVluDKHYQEQ~QEYKQEQEERQK'~QEQLE--RKYQREVEKRYQEQLQKQQQLET
MB
SKQDIGALKQELAKKDEGM(VSEASRKCLRRDLDASREAKKQVEKDLANLTAELDKVKE
. . . . . . .. .. . . . . . . . . . . . . . . . .. . . . . . . . ... . . . . . . . . . 220
I20 PrpM49
23 0 130
240
EKQISEASRKSLSRDLEASREAKKKVEADL
........................... MB
D. PepM49/114-143
M8/276-304
.x
EKQISDASRQGLRRDLDASREAKKQVEKAL 280
M8/233-282
260
140
290
300
200
270
Conserved Conformational Designof Streptococcal M Proteins
5078
a
5
0
0
m
-
0.
-
-5000
0
0
- 10000
t
-15000 -20000
-25000
-
b
c
d
e
f
g
1
L y s Val Lys Glu
5
Val G l u Ala Ala G l u Asn Asn
Val Ala
12
Val S e rS e r
19
C l u LysGluLeuTyr
26
I l e Ala A s p Leu T h r Asp Lys
Arg Arg Asp C l n
33
Asn
40
I l eG l y
G l yC l uT y rL e u
47
G i n Lys Asn L e uG l uL y s
54
C l u H i s G l nS e r
58
Gin Val Ala
G l u Arg
G l u Leu G l u G l u Arg Leu
Ala A s p Lys H i s
6 5T y r
G l n G l u Gln Ala LysLys
72
His
C l n C l u T y rL y s
79
G i n G l u G l u Arg Gln Lys A s n
86
G.ln G l u Gln Leu G l u Arg Lys
Gln G l u
T 9 3y r
-30000 -
9 4 ‘Gin Arg C l u Val G l uL y s I
200
I
220
I
240
x, nrn FIG. 6. Far UV CD spectra of PepM49 in 10 mM sodium phosphate buffer, pH 7.0, in the absence (curve a)and in the presence (curve b) of 20% trifluoroethanol. 0 represents the ellipticity in degrees. cm2.dmol”.
found to contain 23% helix.In the presence of as little as20% trifluoroethanol, a helix-inducing solvent (38, 39), the CD spectrum of PepM49 became very typical of highly a-helical proteins (curue b ) , and the helicity increased to 78%. Further increase of the trifluoroethanolconcentration to 50% increased the helicity only by another 11%.It is thus apparent that a slight increase in the hydrophobicity of the medium contributes significantly to the stabilization of the a-helical structure of the PepM49 protein. Similar stabilization of the helical structure of peptide analogs of tropomyosin, models for two-stranded a-helical coiled-coils, has been observed in the presence of 80% trifluoroethanol (38).The results of the circular dichroic spectral measurements, therefore, demonstrate the propensity of the PepM49 protein to take up a highly a-helical structure with only a marginal lowering of the hydrophilicity of the medium. Heptad Periodicity in the PepM49 Protein-A heptad distribution in the nonpolar amino acid residues, a characteristic of a-helical coiled-coil proteins such as tropomyosin (40), myosin rod region (41), Escherichia coli lipoprotein (42), and other fibrous proteins (43), was observed earlier within the NHz-terminal 60-residue segment ofthe PepM49 protein (31). The repeating heptapeptide pattern of the a-helical coiledcoil proteins can be represented by the generalized sequence (a-b-c-d-e-f-g),, in which residues at positions u and d are generally nonpolar and form a closely packed interface between the two strands of the coiled-coil, thus constituting the “core” residues. The inner positions e and g lie next to the core, and are usually occupied by charged residues. The residues in the outer b,c,f positions, which are mostly polar, point away from the core and arefree to interact with the surrounding molecules (40). Examination of the complete amino acid sequence of the PepM49 molecule has now revealed that the repeating heptad periodicity is present throughout its entire length, but for two interruptions (Fig. 71, an incomplete heptad beginning at Glu-54 and a reversal of a,d phasing at Tyr-93 resulting in a u,d,u,u,d arrangement of the core residues rather than theusual u,d,u,d arrangement. Thus,like the other M molecules, the majority of the PepM49 molecule can adopt a coiled-coil conformation. The interruptions seen in the heptad phasing of the PepM49 protein are not unique to
I01
T y r GlnGluGln
108
G l n Gln Gin
115Lys 122
Leu C l u T h rG l u
Gln I l e S e r G l u Ala S e r
Arg LysSerLeuSer
129Leu
Arg
Leu Gln Lys
Glu
136
LysLysLys
143
Leu
Ala
Arg A s p
S e r Arg G l u Ala
Val G l u Ala A s p
FIG. 7. Heptad periodicity in PepM49 protein. Positions of residues within each period are indicated by letters a-g (4). The three structurally distinct domains within the molecule are indicated by the vertical bars on the right.
this molecule, but have been seen in other M proteins (18, 44), as well as in other coiled-coil proteins (41, 43, 45). As suggested for these proteins, such interruptions in the heptad phasing may cause a localized alteration of the degree of twist of the coiled-coil. Such weak spots could conceivably serve as molecular hinges in thecoiled-coil rod. The pattern of distribution of the nonpolar residues, in conjunction with internal homology, as well as homology with other M proteins, mainly M6, divides the PepM49 protein into three distinct domains, I, 11, and 111. Domain I of PepM49 is comprised of residues 1-46. This region has a very regular distribution of the nonpolar residues at positions u and d. Eighty-five percent of the core u and d positions are occupied by nonpolar residues, the average occupancy seen in fibrous proteins (46). The distribution is less regular in domain I1 (residues 47-114), the nonpolar residues occupying 43% of the core u,d positions. Also, Gln occupies 38% of these positions. Although rare, glutamine has been observed in the u,d positions in other coiled-coil proteins (41, 45, 46), but not in either PepM5 (18) or in M6 (44). Furthermore, 90% of the glutamines and all the three histidines of the molecule are present in this domain. Domain I11 is comprised of residues 115-143. In this domain, 44% of the core u,d positions are occupied by nonpolar and 33% by basic residues, all of latter being in theu positions. Basic residues in thea position have been observed previously in other coiled-coil proteins (46). The higher content of nonpolar residues in the u,d positions within domain I of the PepM49 molecule is suggestive of this domain being more stable than domains I1 and 111. The net charge on the PepM49 protein is essentially neutral. However, the distribution of the acidic and the basic residues within the molecule is not uniform. The acidic as well as the basic residues in the outer b,c,f positions are clustered together. There is a band of negative charges in domain I, essentially at theNHz-terminal endof the molecule. In segment 21-46, 73% of the b,c,f positions are occupied by acidic residues andthere is a complete absence of basic
Conserved Conformational Design of Streptococcal M Proteins
5079
protein serotypes may point to a conformational similarity in this region of the respective native molecules. The far UV CD spectral studies of the PepM49 protein, in conjunction with the heptad periodicity of its nonpolar amino acids, is strongly supportive of the propensity of the molecule to take upan a-helical coiled-coil conformation. The sensitivity of the a-helicity of the PepM49 molecule to slight changes in the hydrophobicity of the environment suggests that in its native state, the stability of the coiled-coil conformation of the NHZ-terminaldomain of the M49 molecule may be modulated by the microenvironment of the bacteria as well as its DISCUSSION COOH-terminal domain and may have significance for the It has long been recognized that antigenic variation of the antiphagocytic property of the molecule. The sequence homology regions of the PepM49 protein Mprotein,through immunological pressure, is a possible means by which the group A streptococcus is able to survive virtually define the boundaries of the three structurally disin the human, its natural host (9). The data seen here, in tinct domains of the molecule. Similar structurally distinct conjunction with the earlier studies (17, 19, 22, 30, 31, 48), domains have been seen in the PepM5 (18) and M6 (44) clearly indicate considerable divergence in the amino acid proteins. While the existence of structurally distinctdomains sequences of the various M protein serotypes. Despite this appears to be a common characteristic of the M proteins, the divergence, varying degrees of conservation among these se- pattern of heptad periodicity in the NHz-terminal region of PepM49, corresponding to its domain I, is clearly distinct quences is also apparent (13, 15, 16, 24-26). A common feature of the rheumatic fever-associated M from that present indomain A of PepM5 andthe correspondprotein serotypes 5, 6, and 24, is the presence of distinct sets ing domain of M6, but similar to that seen in the NHzof identical sequence repeats, the sizes of which vary from terminal region of PepM1, another nephritis-associated serone M protein to another (15, 17, 19, 21, 22). Such identical otype (31). Asn in a positions has been found to be a unique repeats are absent in the PepM49 protein. Nevertheless, the feature in domain A of PepM5 (18) and the corresponding molecule exhibits significant internal homology as seen by domain of M6 (44). A similar trend is seen in the PepM24 dot matrix analysis. To date, M6 is the only M protein for protein (16, 52). However, Asn is present only in one of the a which the completq amino acid sequence is known (19). positions in domain I of the PepM49 protein, and completely absent in the corresponding region of PepMl (31). The presTherefore, if it is considered as a prototypical M protein ence of Gln in the a,d position in domain I1 of PepM49 points molecule, then based on the sequence homology results presented here, the PepM49 protein may also be divided into 3 to differences in the pattern of heptad periodicity in yet distinct regions, A, B, and C, like M6 protein. Regions A and another domain of this molecule, relative to the M5 and M6 B are visualized by the dot matrix plot and region C by its proteins. The PepM49 protein studied here does not have a nonhelhomology with the C-repeat of M6. ical NHz-terminal domain as seen in the M5, M6, and M24 The mechanism by which antigenic variations occur among the different M protein serotypes is not fully understood at proteins (16, 17). Nevertheless, the opsonic antibody epitope present. Size variation among serotypes, as well as among is preserved in the molecule (31).Whether ornot the presence strains of a given serotype has been observed and appears to of a nonhelical NHz-terminal domain is an essential feature play a role in antigenic variation (49, 50). In the case of the of the M protein remains to be investigated further. The present study, in conjunction with earlier studies (16, M6 protein, which contains three distinct sequence repeat regions, A, B, and C (19), homologous recombination events 18, 31, 44), establishes a generally conserved conformational involving intragenic tandem repeatshave been shown to result design for the various M protein serotypes despite considerin changes in size (50, 51), sequence (51), and conformation able variability in the protein sequence. This can explain a (44) of the molecule. In the A repeat region of the M6 common biological function as well as immunological divermolecule, consisting of 5 tandemrepeats, the 3 central repeats sity. Whether the distinct differences observed in the pattern of heptad periodicity in the complete sequence of the PepM49 are identical, while the 2 external repeats contain substitutions (19). Hence, M6 deletion mutations generated by re- protein and the similar trend seen in the partial sequence of moval of central repeatblocks may result in the juxtaposition the PepMl molecule, relative to the PepM5 and M6 proteins of the two external nonidentical repeats (51). The sequence are unique to these two serotypes or a general characteristic repeats in the PepM49 protein are more degenerate than in of other nephritis-associated M protein serotypes is not clear the M5, M6, and M24 proteins. The inexact repeats in the B as yet. Nevertheless, an elucidation of structure-function reregion of PepM49 may have arisen as a result of homologous lationships in the M protein molecule may be expected to of the molecular basis of recombination events of the kind observed in the M6 molecule lead to abetterunderstanding (51). This may also explain the smaller size of PepM49 in streptococcal diseases such as rheumatic fever and glomerurelation to thepepsin fragments of M5 (17), M6 (15, 19), and lonephritis. M24 (21) proteins. The C-repeat of the M6 molecule is immediately adjacent to andon the COOH-terminal side of the pepsin-cleavage site Acknowledgments-We wish to thank Dr. Emil C. Gotschlich for (13, 15, 25). Thus, it is likely that region C of the PepM49 his encouragement and continued interest in these studies, and Drs. protein seen here on the NHz-terminal side of the pepsin Vincent A. Fischetti and Kevin F. Jones for their valuable comments cleavage site could be repeated again on the COOH-terminal on the manuscript. We also wish to thank Drs. Winona C. Barker side of this site inthe native M49 molecule.Extensive identity and Kathryn E. Sidman of the Protein Identification Resource, Biomedical Research Foundation, Georgetown University in the sequence on the NH2-terminal side of the pepsin National Medical Center, Washington, D. C., for performing the dot matrix cleavage site has been seen for the M5 and M6 proteins (15, analysis. The help from Francis Picart in using the CD instrument 44). As suggested earlier (15), the extensive identity in the and the assistance of Y. J. Cho in peptide hydrolyses experiments are sequences around the pepsin cleavage site of the different M acknowledged. residues in these positions. Immediately adjacent is segment 47-71 with basic residue clustering (45% of the b,c,f positions are basic residues, with only 1 acidic residue, Glu). Such alternating bandsof negative and positive charges may permit side to side interaction between molecules. A net negative charge in the NHz-terminal domain of the coiled-coil was reported earlier forthe PepM5 protein(18)and hasbeen seen more recently in the M6 protein (44). This was suggested to play a role in the antiphagocytic property of the M molecule (16, 18, 47).
5080
Conserved Conformational Design of Streptococcal M Proteins REFERENCES
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28. Beachey, E. H., Stollerman, G. H., Chiang, E. Y., Chiang, T. M., Seyer, J. M., and Kang, A. H. (1977)J. Exp. Med. 145, 14691483 29. Beachey, E. H., and Ofek, I. (1976)J. Exp. Med. 143, 759-771 30. Moravek, L., Kuhnemund, O., Havlicek, J., Kopecky, P., and Pavlik, M. (1986)FEBS Lett. 208,435-438 31. Khandke, K. M., Fairwell, T., and Manjula, B. N. (1987)J. Exp. Med. 166,151-162 32. Acharya, A. S., Sussman, L.G., and Manjula, B. N. (1984)J. Chromutogr. 297,37-48 33. Geisler, N., Plessmann, U., and Weber, K. (1985)FEBS Lett. 182,475-478 34. Kirchhausen, T., Scarmato, P., Harrison, S. C., Monroe, J. J., Chow, E. P., Mattaliano, R. J., Ramachandran, K.L., and Smart, J. E., Ahn, A. H., and Brosius, J. (1987)Science 236, 320-324 35. Maizel, J. V., and Zenk, R. P. (1981)Proc. Natl. Acad. Sci. U. S. A. 78,7665-7669 36. Lipman, D. J., and Pearson, W. R. (1985)Science 227, 14351441 37. Garnier, J., Osguthorpe, D. J., and Robson, B. (1978)J. Mol. Biol. 120, 97-120 38. Hodges, R. S., Saund, A. K., Chong, P. C. S., St.-Pierre, S. A., and Reid, R. E. (1981)J. Biol. Chem. 256, 1214-1224 39. Iyer, K. S., and Acharya, A. S. (1987)Proc. Natl. Acad. Sci. U. S. A. 84,7014-7018 40. McLachlan, A. D., and Stewart, M. (1975)J.Mol. Biol. 98, 293304 41. McLachlan, A. D., and Karn, J. (1982)Nature 299,226-231 42. McLachlan, A.D. (1978)J. Mol. Biol. 121,493-506 43. Parry, D.A.D. (1979)in Fibrous Proteins: Scientific, Industrial, and Medical Aspects(Parry, D. A. D., and Creamer, L. K., eds) pp. 393-427,Academic Press, Orlando, FL 44. Fischetti, V. A., Parry, D. A. D., Trus, B. L., Hollingshead, S. K., Scott, J . R., and Manjula, B. N. (1988)Proteins: Structure, Function, and Genetics, Vol. 4,in press 45. Steinert, P. M., Rice, R. H., Roop, D. R., Trus, B. L., and Steven, A. C. (1983)Nature 302, 794-800 46. Parry, D.A. D. (1982)Biosci. Rep. 2,1017-1024 47. Fischetti, V.A. (1983)J. Immunol. 130,896-902 48. Seyer, J. M., Kang, A. H., and Beachey, E. H. (1980)Biochem. Biophys. Res. Commun. 92,546-553 49. Fischetti, V. A., Jones, K. F., and Scott, J. R. (1985)J.Exp. Med. 161,1384-1401 50. Fischetti, V. A., Jarymowycz, M., Jones, K. F., and Scott, J. R. (1986)J. Exp. Med. 164,971-980 51. Hollingshead, S. K., Fischetti, V. A., and Scott, J. R. (1987)Mol. Gen. Genet. 207,196-203 52. Fischetti, V.A., and Manjula, B. N. (1982) in Seminars in Infectious Diseases: Bacterial Vaccines (Robbins, J. B., Hill, J. C., and Sadoff, J. C., eds) Vol.IV, pp. 411-418, ThiemeStratton, Inc., New York 53. Dayhoff, M. O., Barker, W. C., and Hunt, L. P. (1983)Methods Enzymol. 91,524-545 54. Zimmerman, C.L., Appella, E., and Pisano, J. J. (1977)Anal. Biochem. 77,569-573 55. Fairwell, T. (1983)Methods Enzymol. 91,502-511 56. Chang, C. T., Wu, C. C., and Yang, J. T. (1978)Anal. Biochem. 91, 13-31 Continued on next page.
Conserved Conformational Design of Streptococcal M Proteins
generally obrained.
:I
f
Ulrn .Cld
5081
5082
Conserved Conformational Designof Streptococcal M Proteins Table 2 S e w e n s r data on
Table 3
che frypflc peptides of DHP-PepMllP GlU 8 7
Lyr Nq
Lyr NQ
Tyr Cln 9.1 3 2
Arg
LYl NQ
ser
Le"
ser Arg
4.2
6 7
3.0
Cl" V S l 9.6 9 4
Arg
3 6
1.3 2 i
Le" Cl" Ala s e x 15.8 17 9 13.6 12.1 2 . 8
A'P
dl" Lyr 13.7 NQ
AS"
M i n o Acld
Cl"
Gl"
Acg
1.9 Cl"
Le"
21.6 16.9 11.1 1 3 . 9 8 6
Cl"
A'&
10.1 2 9
Clu Ala LysLyrLys 13.3 10.3 NQ NQ
NQ
Glu 75
&la Asp Leu 8.5 5 0 0.85
Cl" 1 6 5 1 0 . 1 5.6
Gl"
Lys
4.L
k" Cl" 7 I 6.7
Gl" 1.9
Thr Clu 1.3 0.6 3 . 3
Lys NQ
Cln 11e 3 8 4.7
Tyr
Cl"
Glu
Cln Lys 15 2 NQ
Val
U" Cl" Ly' 1 3 . 3 I & . &15.8 NQ
AS"
Ala A l a ASP Lys 10.9 13.7 13.9 10 8 NQ
Vel
Lys
NQ
HI. 3.9
9.9
Cln 4.2
Clu 3.8
HIS 6 3
TyrLys 3 . 1 NQ
Cl" 5.5
Cln 5.7
Leu 3.2
Ser 0.8
Clu A l a
Ser 0 5
Arl 0.5
Le"
Cl"
Cln ser 1 0 . 8 2 0 . 8 12.5 8.6 Ng
Cl" 7.1
Tyr 8.3
Gln CLu Cln 2.9 7.5 6 . 6
Lya NQ
dln 2.2
Clu 2 2
Ng
2 .36. 6 "IS
Ala 6 8
Oln Glu Clu Azg I 2 2.1 3.1 NQ
Composition of the Chp0,rryptlc peptides
of
Peplli9
