8158
Biochemistry 1987, 26, 8 158-8 165
Levin, I. W. (1 984) in Advances of Infrared and Raman Specfroscopy (Clark, R. J. H., & Hester, R. E., Eds.) Vol. 11, Wiley-Heyden, London. Levin, I., Lavialle, F., & Mollay, C. (1982) Biophys. J . 37, 339-349. Mendelsohn, R., & Mantsch, H. H. (1986) in Progress in Protein-Lipid Interactions, 2nd ed. (Watts, A., & De Pont, J. J. H. H. M., Eds.) pp 103-146, Elsevier, Amsterdam. Mendelsohn, R., Dluhy, R., Taraschi, T., Cameron, D. G., & Mantsch, H. H. M. (1981) Biochemistry 20, 6699-6706. Mollay, C., Kreil, G., & Berger, H. (1976) Biochim. Biophys. Acta 426, 317-324. Okamura, E., Umemura, J., & Takenaka, T. (1986) Biochim. Biophys. Acta 856, 68-75. Olson, F. C., Munjal, D., & Malviya, A. N. (1974) Toxicon 12, 419-425.
Pincus, M. R., Klausner, R. D., & Scheraga, H. A. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 5107-5111. Snyder, R. G., Hsu, S.L., & Krimm, S. (1978) Spectrochim. Acta, Part A 34A, 395-406. Terwilliger, T. C., Weissman, L., & Eisenberg, D. (1982) Biophys. J . 37, 353-361. Verma, S. P., Wallach, D. F. H., & Smith, I. C. P. (1974) Biochim. Biophys. Acta 345, 129-140. Vogel, H., & Jahnig, F. (1986) Biophys. J . 50, 573-582. Vogel, H., Jahnig, F., Hoffmann, V., & Stumpel, J. (1983) Biochim. Biophys. Acta 733, 201-209. Weissmann, G., Hirschlorn, R., & Kreakaver, K. (1969) Biochem. Pharmacol. 18, 1771-1775. Yunes, R., Goldhammer, A. R., Garner, W. K., & Cordes, E. H. (1977) Arch. Biochem. Biophys. 183, 105-112.
Comparison of cDNA-Derived Protein Sequences of the Human Fibronectin and Vitronectin Receptor a-Subunits and Platelet Glycoprotein IIbt Laurence A. Fitzgerald,*.* Mortimer Poncz,s Beat Steiner,fll Stanley C. Rall, Jr.,t Joel S. Bennett,l and David R. Phillips$ Gladstone Foundation Laboratories for Cardiovascular Disease, Cardiovascular Research Institute, Department of Pathology, University of California, San Francisco, California 94140-0608, Division of Hematology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, and The Hospital of the University of Pennsylvania, Department of Medicine, The University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 Received June 10, 1987; Revised Manuscript Received August 4, 1987
ABSTRACT: The fibronectin receptor (FnR), the vitronectin receptor (VnR), and the platelet membrane glycoprotein (GP) IIb-IIIa complex are members of a family of cell adhesion receptors, which consist of noncovalently associated a-and @-subunits. The present study was designed to compare the cDNA-derived protein sequences of the a-subunits of human FnR, VnR, and platelet G P IIb. cDNA clones for the a-subunit of the FnR (FnR,) were obtained from a human umbilical vein endothelial (HUVE) cell library by using an oligonucleotide probe designed from a peptide sequence of platelet G P IIb. cDNA clones for platelet G P IIb were isolated from a cDNA expression library of human erythroleukemia cells by using antibodies. cDNA clones of the VnR a-subunit (VnR,) were obtained from the HUVE cell library by using an oligonucleotideprobe from the partial cDNA sequence for the VnR,. Translation of these sequences showed that the FNR,, the VnR,, and G P IIb are composed of disulfide-linked large (858-871 amino acids) and small (1 37-1 58 amino acids) chains that are posttranslationally processed from a single mRNA. A single hydrophobic segment located near the carboxyl terminus of each small chain appears to be a transmembrane domain. The large chains appear to be entirely extracellular, and each contains four repeated putative Ca2+-bindingdomains of about 30 amino acids that have sequence similarities to other Ca2+-bindingproteins. The identity among the protein sequences of the three receptor a-subunits ranges from 36.1% to 44.5%, with the Ca2+-bindingdomains having the greatest homology. These proteins apparently evolved by a process of gene duplication.
C e l l adhesion and platelet aggregation are mediated by the binding of adhesive ligands, such as fibronectin and fibrinogen, to a family of cell membrane receptors (Ruoslahti & Pierschbacher, 1986; Hynes, 1987). These adhesion receptors 'This research was supported by National Institutes of Health Grants HL 28947 and HL 32254 (to D.R.P.). *Correspondence should be addressed to this author at the Gladstone Foundation Laboratories, San Francisco, CA 94140-0608. *University of California, San Francisco. *The Children's Hospital of Philadelphia. Present address: Hoffmann-La Roche, Pharmaceutical Research, CH-4002 Basel, Switzerland. The University of Pennsylvania School of Medicine.
0006-2960/87/0426-8 158%01S O / O
consist of heterodimer complexes of noncovalently linked a-subunits ( M , 140 000-200 000) and @-subunits ( M , 95000-120000). Although there are at least 10 receptors in this family, there are only 3 known @-subunits,each of which defines a subfamily of receptors (Hynes, 1987). One subfamily consists of the platelet glycoprotein (GP)'IIb-IIIa complex, which is required for platelet aggregation, and the widely
'
Abbreviations: GP, glycoprotein; VnR, vitronectin receptor; FnR, fibronectin receptor; FnR,, fibronectin receptor a-subunit; VLA, very late antigens; SDS, sodium dodecyl sulfate; VnR,, vitronectin receptor asubunit; HUVE cell, human umbilical vein endothelial cell; HEL cell, human erythroleukemia cell; SSC, saline-sodium citrate; kDa, kilodalton(s); Tris-HCl, tris(hydroxymethy1)aminomethane hydrochloride.
0 1987 American Chemical Society
ADHESION RECEPTOR &-SUBUNITS
VOL. 26, NO. 2 5 , 1987
8159
distributed vitronectin receptor (VnR); they share GP IIIa as their 8-subunit (Ginsberg et al., 1987). A second subfamily includes the widely distributed human fibronectin receptor (FnRtand the very late antigens (VLA) (first described on stimulated T-lymphocytes), which share a 120-kDa &subunit (Hemler et al., 1987; Takada et al., 1987a). The five asubunits in the mammalian FnR/VLA receptor subfamily are distinct proteins, with the FnR a-subunit (FnR,) having a molecular weight of 160 000 on nonreduced sodium dodecyl sulfate (SDS)-polyacrylamide gels. In chickens, there appear to be two separate FnR a-subunits, which have different molecular weights (band 1, MI 160 000; band 2, MI 140 000) and are noncovalently complexed to a 8-subunit of MI 120000 (band 3) (Buck et al., 1985, 1986). A third adhesion receptor subfamily consists of the leukocyte Mac-1, LFA-1, and p150/95 antigens (Springer & Anderson, 1985), which share a 95-kDa @subunit (Kishimoto et al., 1987). The receptors in this third subfamily mediate the interactions of leukocytes with endothelial cells and with the C3bi fragment of complement (Micklem & Sim, 1985; Springer & Anderson, 1985; Kishimoto et al., 1987). The cDNA and deduced protein sequences for each of these adhesion receptor IS-subunits have been determined (Tamkun et al., 1986; Fitzgerald et al., 1987; Kishimoto et al., 1987). These proteins share a number of structural features, including conservation of all 56 cysteines. The P-subunit protein sequences are homologous (38545.4% of the amino acids are identical), indicating that they probably evolved by a process of gene duplication.2 The overall protein structure and sequence homology among the a-subunits in the adhesion receptor family are less well understood. These proteins are diverse in size, and some consist of two-chain, disulfide-linked polypeptides (e.g., GP IIb) (Argraves et al., 1986; Suzuki et al., 1986; Hynes, 1987; Poncz et al., 1987). Partial amino-terminal sequences of several a-subunits (GP IIb, VnR,, VLA, LFA-1, and Mac-1) are -40% identical (Charo et al., 1986; Suzuki et al., 1986; Takada et al., 1987b). Partial cDNA sequences for the human FnR, (Argraves et al., 1986) and VnR, have been reported, and the carboxyl-terminal cDNA-deduced protein sequences are -40% identical. This report describes the isolation of full-length human endothelial cell cDNA clones for the FnR, and of cDNA clones and the sequence corresponding to the amino-terminal region of the VnR,. The complete deduced protein sequence of the FnR, aligns with the cDNA-derived protein sequences for platelet GP IIb and the VnR,. These three a-subunits have a number of common structural features, including the presence of four repeated putative Ca*+-bindingdomains and a similar positioning of the transmembrane segment. The overall protein sequence homology suggests that they, too, evolved by a process of separate gene duplication.
peptides were dansylated (Stephens, 1975), mixed with the remaining peptides, and fractionated by electrophoresis through a 10-18% SDS-polyacrylamide gel (Laemmli, 1970). Peptide fragments were visualized with UV light and electroeluted from the gel slices. Protein sequences were determined from the amino terminus of the intact GP IIb large and small chains and from three cyanogen bromide fragments of the large chain (approximately 2 nmol of each) using a Beckman 890M sequencer (Beckman Instruments, Fullerton, CA) (Weisgraber et al., 1981). ( B ) Preparation of Oligonucleotide Probes. The GP IIb peptide sequences were used to design oligonucleotide probes according to principles of codon use described by Lathe (1985). Probes 42-5 1 nucleotides in length were synthesized by using an Applied Biosystems (Foster City, CA) 380B synthesizer. The oligonucleotides were purified by electrophoresis through 10% polyacrylamide gels containing 7 M urea and were recovered from gel slices. The probes were labeled on the 5' end using [ Y - ~ ~ P I A (New T P England Nuclear, Boston, MA) and T4 polynucleotide kinase (Boehringer Mannheim, Indianapolis, IN). (C) Screening the cDNA Library. A randomly primed human umbilical vein endothelial (HUVE) cell cDNA library was prepared in Xgtll as described (Young & Davis, 1983; Ginsberg et al., 1985). Plaques were screened with the Escherichia coli Y 1088 strain by hybridization of nitrocellulose filters in 20% formamide, 5 X SSC (SSC is 150 mM NaCl/ 15 mM sodium citrate), 5 X Denhardt's, 0.1% SDS, and 0.2 mg/mL E. coli tRNA at 42 OC (Maniatis et al., 1982). Filters were washed using 2 X SSC at 42-45 "C. Positive plaques were purified and the X cDNA inserts subcloned into pUC vectors for restriction analysis. The cDNA restriction fragments were subcloned into M13 vectors, and the DNA was sequenced by the dideoxy method of Sanger et al. (1977) using ~ A T P - ~ ' - [ ( Y -thiophosphate ~~S] (New England Nuclear). Sequence data analysis was performed by using the BIONET system of Intelligenetics, Inc. (Palo Alto, CA). Isolation of GP IIb cDNAs. cDNA clones specific for GP IIb were isolated from a human erythroleukemia (HEL) cell cDNA Xgtll expression library as described (Poncz et al., 1987). Isolation of VnR, cDNAs. The published partial cDNA sequence of VnR, was used to obtain cDNA clones for the VnR,. A 42-mer oligonucleotide probe corresponding to nucleotides 78-119 of this sequence (Suzuki et al., 1986) was synthesized, purified, 32P-labeledon the 5' end, and used to screen the randomly primed HUVE cell cDNA library as described above, except that the filters were washed with 2 X SSC at 50-55 "C. Only the 5' 1323 nucleotides of a VnR, cDNA clone were sequenced, which included the first Hind111 site in the published sequence.
EXPERIMENTAL PROCEDURES Isolation of FnR a-Subunit c ~ N A s .( A ) Preparation of GP IIb Peptides and Protein Sequencing. Both the large and small chains of GP IIb were isolated by electroelution from preparative SDS gels of the purified platelet GP IIb-IIIa complex (Fitzgerald et al., 1985a). For cyanogen bromide cleavage, 2.5 mg (30 nmol) of protein was dissolved in 3.3 mL of 70% formic acid and treated with 326 mg of cyanogen bromide for 2 days at ambient temperature. The peptides were first dialyzed against 50% formic acid and then against 0.2 M Tris.HCl/l% SDS, pH 7.87. Ten percent of the dialyzed
RESULTS The FnR, cDNA clones were isolated from a HUVE cell cDNA library by using an oligonucleotideprobe designed from the platelet G P IIb peptide sequence. Figure 1A shows the sequence of this probe, which matched the cDNA sequence in 40 of 51 positions, of which the first 18 nucleotides were correctly assigned. A translation of the cDNA sequence matched the sequence of the platelet GP IIb peptide in 14 of 17 positions, with two 6-residue stretches of exact identity (Figure 1B). Other oligonucleotideprobes designed from GP IIb peptides did not detect positive plaques in the HUVE cell cDNA library. While this work was in progress, a partial cDNA sequence was reported for the carboxyl-terminal region of FnR, (Argraves et al., 1986). The published sequence is
L. Fitzgerald and D. R. Phillips, unpublished observations.
8160
BIOCHEMISTRY
A.
R.
F I T Z G E R A L D ET A L .
Oligonucleotide probe
ATG GCC TCC TAC TTT GGC CAC TCT GTG GCT GTG ACA GAT GTG AAT GGC GAT
cDNA sequence
ATG GCC TCC TAC TTT GGC TAT GCA GTG GCC GCC ACA GAC GTC AAT GGG GAC
GP IIb peptide sequence
(tl)
cDNA translated sequence
M
I IIIII I
111111111111111111 I A
S
Y
F
S
Y
F
G
S
Ii
V
I I I I I II I I I I I II
V
A
D
T
V
N
G
0
FIGURE 1: Selection of FnR, cDNA clones using an oligonucleotideprobe designed from a peptide of platelet GP IIb. (A) Nucleotide sequence of the 51-mer oligonucleotide probe vs the FnR, cDNA nucleotide sequence. (B) Amino-terminal protein sequence of the GP IIb cyanogen bromide fragment vs the translation of the probe-selected cDNA clone. Identities are indicated by vertical bars. The methionine (M) in the peptide sequence was deduced from cyanogen bromide cleavage.
- - -----
7 Pst I
EcoRI
P s t I Fco R I
PstI
PstI
Sac I Sac I Bam H I (
911
Pst I
PstI
Hindm
Bam H I
5'
3'
Clone1-3 I
I
I
I
Clone 1-7 I
i
lkb FIGURE
2: Diagram of the FnR, cDNA clones (1-3, 1-7) showing relevant restriction sites and the sequencing strategy. CGCTGGGGCCCCCGGCGCCGACCCCCGCTGCTGCCGCTGCTGTTGCTGCTGCTGCCGCCGCCACCCffiGGTCGGGGGC R Y G P R R R P P L L P L L L L L L P P P P R V G G
-26
TTCMCTTAWCGCGGAGGCCCCAGCffiTACTCTCffiGGCCCCCGGGCTCCTTCTTCGGATTCTCffiTGGAGTTTTACCGGCCGGGMCA F W L D A E A P A V L S G P P G S F F G F S V E F Y R P G T
I
18
AAGCAGAAGS~AGGGGTACGGCGGGCACTGTTCCTGGCCTCCAGGCAffiCMCCCTGACCCAGACCCTGCTCATCCAGMT~GCTCGA 1698 S I I K Q K G G V R R A L f L A S R Q A T L T Q T L L I Q N G A R
168
GAGGATTGCAWIGAGATGMGATCTACCTCAGGMCGAGTCAGMTTTCGffiACMACTCTCGCCGATTCACATCGCTCTCMCTTCICC 1 E D C R E ~ K l Y L R N E S E f R D K L S P l ~ l A l
1788 ~ f S
5
4
3
G A C G C G G T C A G T G T G C T G G T G G G A G C A C C C M G G C T M T A C 258 1 O G V S V L V G A P K A N T S Q P G V L O G G A V Y L C P U
5
T T G W C C C C C A A G C C C C A G T G G A C A G C C A C G G C C t T C A C T C A W I T C 7 1 L D P Q A P V D S H G L R P A L M Y Q S K S R l f D K A Q l
6
GGTGCCAGCCCCMAtAGTGCACCCCCATTGMTTTGACAGC~CTCTCGGCTCCTffiAGTCCTCACTGTCCffiCTC~A~AGAG 348 1 G A S P T Q C T P I E F D S K G S R L L E S S L S S S E G E
~
TTGCTGGACTGTGGAGMGACMCATCTGTGTGCCTGACCTGCAGCTGGMGTGTTTG~ffiCffiMCCATGTGTACCT~TWIC~ 1968 O I L L D C C E D N I C V P D L ~ L E V F G E ~ N ~ V ~ L
9
WIGCCTGTffiffiTACMGTCCTTGCffiTffiTTC~GCMCffiTTCGAGCCCATGGCTCCTCCATCTTffiCAT6CGCTCCACT6TACAGC l E P V E V K S L Q U F G A T V R A H G S S l L A C A P L V S
6
3
MTGCCCTGMCCTCACTTTCCATGCCCAGMTGTGGGTGffiGGTGGCGCCTATGffiGCT~CTTCGGGTCAC~CCCTCCAGAffiCT 1 N A L N L T f H A Q N V G E G G A Y E A E L R V T A P P E A
6
6
G~~~TACTCAGGACTCGTCAGACACCCA~~~GMCTTCTCCAGCCTGAGCTGTCACTACTTTGCCGT~CCAWIGCCGCCTGCT~~~TGTGT 2148 1 E V S G L V R H P G N F S S L S C D Y F A V N Q S R L L V C
6
9
WICCTGGGCAACCCCATGMGGCAGGAGCCAGTCTGTGGGGTGGCCTTCGGTTTACAGTCCCTCATCTCCffiGACACT~MAACC~TC 1 D L G N P H K A G A S L Y G G L R F T V P M L R D T K K T I
1
A
2
438
TGGCGCACAWIGMGGAGCCACTGAGCCACCCCGTffiGCACCTGCTACCTCTCCAC~TMCTTCACCCGMTTCTG~TATGCACCC 528 1 Y R T E K E P L S D P V G T C V L S T D N F T R l L ~ Y A P
A
T G C C G C T C f f i A T T T C f f i C T G G G C f f i C f f i C A C A G G G T . T # T G 1 5 l C R S D F S Y A A G Q G Y C Q G G F S A E F T K T G R V V L
618
A
A
1818
A
2238
C A G T T T G A C T T C C A G A T C C T C A G C M G A A T C T ~ C M C T C G C ~ G C G A C G T G G T T T C C T T T C ~ T C T C C G T ~ G C T C f f i G C C C f f2328 i 1 2 1 Q F D F Q I L S K N L N N S Q S D V V S F R L S V E A Q A Q
AACCTffi~CAGGGGCAGCTGCACACTCGCCffiGCCffiTTCCATCTATGATWICAGCTACCT~ATACTCTGTGGCTG~ffiTGMTTC 798 2 l l N L V Q G Q L Q T R Q A S S l Y D D S Y L G V S V A V G E F
7
5
GTCACCCTGMCGGTGTCTCCMGCCTGAGGCAGTGCTATTCCCAGTMGCGACTGGCATCCCCWIGACC~~~CCTC~U~~~~~C 2418 1 V T L N G V S K P E A V L F P V S D Y M P R D Q P Q K E E D
A G T f f i T W T G A C A C A G M G M T T T G ~ G C T f f i T G T G C C C C M A G A C A T T 888 2 4 1 S G D D T E D F V A G V P K 6 M L T V 6 Y V T l L N G S D l
7
8
CTGGGACCTGCTGTCCACCATGTCTATGAGCTCATCMCCMGGCCCCAGCTCCATTAGCCAGGGTGTGCTffiMCTC~TGTCCCCffi 1 L G P A V H H V V E L I N Q G P S S l S Q G V L E l S C P
A
D
A
GGTGGACCAGWAGCTATTTCTGGCMGGCCffiATCCTGTCTGCCACTC~AGC~TTGCffiMTCTTATTACCCCGffiTA~TGATC708 1 8 l G G P G S Y F Y Q G Q I l S A T Q E Q I A E S Y V P E V L l
A
G
2058
A
2508 Q
C G A T C C C T C T A C M C T T C T C ~ t A G A T ~ C T C C T A C T T T f f i C T A T ~ f f i T 6 G C C ~ C M ~ C G T ~ T ~ C ~ 9C7T8 G G A T GCTCTGGAffiGTCAGCAGCTCCTATATGTGACCAGAGTTACGGGACTCMCTGCACCACCMTCACCCCATTMCCC~ffiGCCT~ 2598 2 7 1 R S L V N F S G E Q M A S V F G V A V A A T D V N G D G l D 8 1 1 A L E G Q Q L L Y V T R V T G L N C T T N H P l N P K G l ~
A
A
C A C T T G C T f f i T G U ; f f i ~ C C C C T 6 C T C A T G C A T C G G A C C t C ID68 3 D l D L L V G A P L l M D R T P D G R P Q E V G R V V V Y L Q M
8
CCAGCCGGCATAGAGCCCACGCCCACCCTTMCCTCACTGGCCATGAT~TTT~CCATTTffiCAGCTCCTTGACCCCCCTGGG~C 1158 3 3 1 P A G I E P T P T L T L T G M D E F G R F G S S l T P L G D
CCGGAGGCTGAGTGTTTCAGGCTGCGCTGTGAGCTCGGGCCCCTGCACCMCAAGAGAGCC~GTCTGCffiTTGCATTTCCGAGTCTGG 2 1 1 8 W I P E A E C F R L R C E L G P L H Q Q E S Q S L Q L H F R V N
C T G G A C C A G G A T G G C T A C M T G A T 6 T ~ C A T C ~ C T C C C T T T U i T ~ A C A C C C f f i C ~ T f f i T 6 T T T G T A T T T C C1248 TG~ 3 6 1 L D Q D G V N D V A I 6 A P F G G E T Q Q 6 V V F V F P G G
9
C C ~ G G C T U i G C T C T M G C C T T C C ~ T T C T G C ~ C C C T 6 T ~ C f f i C C A G C C A C A C C C C f f i A C T T C ~ T G G C T C T G C C C T T C G1338 A - 3 9 I P G G L G S K P S Q V L Q P L Y A A S H T P D F F G S A L R GGAGGCCCA~CCTGGATffiCMTGGATATCCTGATCTGATTGT~UiTCCTTTGGTGTGGACMG~TGTffiTATACAGGGGCC~CCC 1428 4 2 1 G G R D L D G N G Y P D L I V G S F G V D K A V V V R G R P ATCGTGTCCGCTffiTGCCTCCCTCACCATCTTCCCCGCCATGTTCMCCtA~AGCffiffiCTGCffiCTTffiffiffi~CCCTGTGGCC 151b 1 I V S A S A S L T I F P A M F I P E E R S C S L E G M P V A
4
5
4
TGCATCMCCTTffiCTTCTGCCTCMTGCTTCTG~CACGTTGCTGACTCCATTUiTTTCACAGTGGMCTTCffiCTffiACTGGCffi 1608 8 1 C I N l S f C L N A S G K H V A D S l G F T V E L Q L D U Q
A
A
4
D
TTGGATCCCGAGGGTTCCCTGCACCACCAGCMAAACGGGMGCTCCMGCCGCAGCTCTGCTTCCTCGGGACCTCffiATCCTG~TMTCC 2688 1 L D P E G S L H H Q Q K R E A P S R S S A S S G P Q l L K C
4
GCCAAGACTTTCTTGCAGCGGGAGCACCAGCCATTTAGCCTGCAGTGTGAGGCTGTGTAC~GCCCTGMGATGCCCTACCWATCCTG 2868 l ~ A K T F l Q R E M Q P F S L Q C E A V V K A L K M P V R l L
CCTCGGCAGCTGCCCCAMAAGAGCGTCAGGTGGCCACAGCTGTGCAATGGACCMffiCAGMGGCAGCTATGGCGTCCCACTGTGGATC 2958 9 3 1 P R Q L P Q K E R Q V A T A V Q Y T K A E G S Y G V P L Y I 9
6
991
ATCATCCTAGCCATCCTGTTTGGCCTCCTGCTCCTAGGTCTACTCATCTACATCCTCTAC~GCTTffiATTCTTC~CGCTCCCTCCCA 3048 1 I I L A I L F G L L L L G L L I Y I L Y K L G f f K R S L P TATGGCACCGCCATGGMAAAGCTCAGCTCAAGCCTCCAGCCACCTCTGATGCCtgagtcctcccaatttcagactcccattcctgaaga 3138 Y G T A H E K A Q L K P P A T S D AStOp
accagtccccccaccctcattctactgaaaaggaggggtctgggtacttcttgaaggtgctgacggccagggagaagctcctctccccag 3220 cccagagacatacttgaagggccagagccaggggggtgaggagctggggatcc 3281
FIGURE 3: FnR, cDNA sequence and protein translation. Amino acids -26 to -1 represent the signal peptide. The putative transmembrane segment is indicated by a solid underline. Potential N-linked glycosylation sites (A)and the junction between the large and small chains (arrow) are indicated. The published nucleotide sequence (Argraves et al., 1986) begins at position 2416. The protein sequence homologous to GP IIb (Figure 1) that was the basis for selecting this clone is indicated by the dashed line.
identical with that of the HUVE cDNA clone for 866 nucleotides at the 3' end. A restriction map and the sequencing strategy used for the two overlapping FnR, cDNA clones are shown in Figure 2. The 328 1-base cDNA sequence was obtained on both strands. Figure 3 shows the cDNA sequence and its translation, which resulted in a single open-reading frame including the entire mature protein (1008 amino acids) and part of the signal peptide (26 amino acids). The cDNG sequence includes only 179 nucleotides of the 3' untranslated region, which has been reported to be 1048 nucleotides long (Argraves et al., 1986). The peptide sequence of the amino terminus of the FnR, has not been reported; it was assigned from the cDNA translation on the basis that the a-subunits of the adhesion receptor family have an almost invariant Asn-Leu-Asp sequence in positions
2-4 (Charo et al., 1986; Suzuki et al., 1986; Takada et al., 1987b). The derived amino acid sequence (Figure 3, -26 to -1) 5' to the predicted amino terminus contains primarily hydrophobic residues, a characteristic of signal peptides. Analysis of the FnR, protein sequence by the method of Kyte and Doolittle (1982) using a windowing average of 15 residues showed a single, strongly hydrophobic stretch from residues 952 through 980 (Figure 3). This region probably represents the transmembrane domain. Thus, over 90% of the FnR, is extracellular,the putative cytoplasmic domain having only 28 residues. The FnR, consists of one large and one small chain, which are disulfide linked (Argraves et al., 1986; Hynes, 1986). Both of these chains are encoded by a single mRNA, and the propeptide is believed to be cleaved between the arginine and serine residues at positions 858 and 859 (Argraves et al., 1986;
K
ADHESION RECEPTOR ff-SUBUNITS
VOL. 26, N O . 2 5 , 1987 C g g t c C C ~ C g c g c ~ C t t C g g C ( j 22
ATffiClTTTCCGCCGCGGCGACGGCTGCGCC~CGGlCCCCGCGGCCTCCCGCTlClTClCTCGG~CTCCTGCTACClCTGlGCCGCGCC 1 1 2 - 3 O M A F P P R R R L R L G P R G L P L L L S G L L L P L C R A TTCMCClAGACGTGGACAGTCClGCCGffilACTCTGGCCCCGAGGGMGTlAClTCGGClTCGCCGlGGATTTCTTCGlGCCCAGCGCG 202 F N L D Y D S P A E Y S G P E G S V F G F A ~ D F F ~ P S A
1
TCTTCCCGGAlGTTTCTTClCGlGGGA~lCCCMAGCAMCACCACCCAGCClGGGAllGlGGAAGGAGGGCAGGlCCTCMATGTGAC 31 S S R U F L L V G A P K A N 1 1 P P G I Y E G G P V L Y C D
292
A
6
lGGlCllCTlCCCGCCGGTGCCAGCCMTlGAATTlGAlGCMCAGGCMllGAGATTAlGCCAAGGAlGAlCCAlTGGMlllAAGlCC 382 1 Y S S l R R C Q P I E F D A l G N R D I A Y D D P L E F K S
~
CAlCAGTGGTlTGGAGCATClGlGAGGlCGAMCAGGAlAMATllTGGCCTGlGCCCCATlGlACCAllGGAGMClGAGAlGAMCAG ~ H ~ Y F G ~ ~ Y R S Y P D K I L A C A
P
GAGCGAGAGCCTGlTGGMCAlGCTlTCllCMGAlffiGACAAAGAClGTlGAGlAlGClCCAlGlAGAlCACAAGATAllGAlGClGAl 121 E R E P V G T C F L P D G T Y T Y E Y A P C R S P O I O A D
412
~
~
562
GGACAGGGATTTTGlCAAGGAGGAllCAGCAllGAllllAClMAGClGACAGAGTACllCTTG~lGGlCClGGlffiCllTlAllGGCAA 652 1 G P G F C Q G G F S l D ~ T K A O R V L L G G P G S F I U ~
1
5
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GGTC~GCTTATTTCGGATCAffiTGGCA~lCGlATCTAMlACGACCCCMlGTlTACAGCAlCAAGlAlAATMCCMllAGCAACl 742 ~ ~ G P L I S D I YP S YV Y A D P LN V I S I Y Y N N Q L A T
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Suzuki et al., 1986). Thus, the FnR, consists of a large chain of 858 amino acids and a small chain of 150 amino acids. The FnR, contains 14 potential N-linked glycosylation sites having the sequence Asn-X-Ser/Thr, all of which are in the large subunit (Figure 3). Thus, the difference between the cDNApredicted molecular weight for the large chain (92 941) and the molecular weight determined from SDS gels (140 000) (Pytela et al., 1986) can be largely accounted for by the number of potential glycosylation sites. The reason for the difference between the cDNA-predicted molecular weight of the small chain (17 088) and that estimated from SDS gels (25 000) is not clear. A HUVE cell cDNA clone specific for the VnR, was obtained by using an oligonucleotide probe made to a region of the recently published partial cDNA sequence for this protein (Suzuki et al., 1986). Figure 4 shows the cDNA sequence and protein translation for the 5' region of the VnR,, which contains the amino-terminal382 amino acids, a putative 29 amino acid signal peptide, and 22 nucleotides of the 5' untranslated sequence, all of which were missing in the published sequence. Sixty-seven nucleotides that overlap the published sequence and a 240-base segment 5' to the first Sac1 site (data not shown) were also identified; they were identical with those of the published sequence. When the cDNA sequence in the present report is combined with the published sequence, the deduced sequence of VnR, contains 1018 amino acids: 860 in the large chain and 158 in the small chain. The VnR, has 13 potential N-linked glycosylation sites: 10 in the large chain and 3 in the small chain. The cDNA-deduced amino terminus of the large chain is identical with the published amino acid sequence (Suzuki et al., 1986). The cDNAs corresponding to platelet G P IIb were isolated from a HEL cell cDNA expression library by antibody screening (Poncz et al., 1987). These cDNAs contain the entire G P IIb protein-coding sequence, including 1008 amino acids for the mature protein and 30 amino acids representing a signal peptide. The G P IIb large chain contains 871 amino acids, and the small chain contains 137 amino acids. The predicted G P IIb protein sequence contains the partial peptide sequences obtained from the amino termini of the large and
~
8161
Table I: Protein Sequence Identities in the Three a-Subunits' small large chain chain overall no. % no. % no. % 27.1 common 261 28.9 27 17.0 288 424 49 30.8 473 44.5 FnR,vs VnR, 47.0 402 37.9 359 39.8 43 27.1 H ~ FnR,vs ~ T E GP ~ IIb Y ~ VnR, vs GP IIb 340 37.7 383 36.1 43 27.0 aCalculated from data shown in Figure 5. To determine percentages, the numbers were derived by the total of positions in the alignment, including gaps.
small chains of purified platelet G P IIb (Charo et al., 1986). In addition, the 17 amino acids used to design the oligo~ nucleotide ~ i ~ ~ ~ probe ~ ~ ~ shown G t f in G Figure C C A ~ ~ contained ~ ~ ~ ~ in ~ ~positions ~ 1C are 285-301 of the translated GP IIb sequence (Figure 5 ) . These sequence identities confirm that the GP IIb like glycoprotein synthesized and expressed by HEL cells is the same gene product as that found in platelets. There are five potential N-linked glycosylation sites in GP IIb: four in the large chain and one in the small chain. An alignment of the cDNA-predicted protein sequences of the large and small chains of the three proteins is shown in Figure 5 . The best alignment was obtained when the cysteines were placed in register. The sizes of the large and small chains in the three proteins differ slightly. Table I lists the overall identities in protein sequence. A majority (288 positions) of the amino acid identities are conserved in all three proteins. The FnR, and VnR, are the most homologous, while G P IIb and the VnR, are the least homologous. The large chains are more homologous than the small chains. The region of greatest similarity among all three a-subunits extends from positions 224-436 (FnR,); 93 identical positions (43.7%) are found in this region. Each protein in this region has four repeated segments (Figure 5 ) of about 30 amino acids. Within each segment is a stretch of 12 amino acids that contains Asp, Asn, and hydrophobic residues in a spacing that resembles the p-turns of other Ca2+-bindingproteins (Kretsinger & Nockolds, 1973; Garigpy & Hodges, 1983). Figure 6 shows a representative Caz+-binding repeat of the FnR, and bovine brain calmodulin. This Ca2+-binding sequence of calmodulin has the characteristic "EF hand" structure: the Ca2+-binding coordinates are located in a 0-turn, which is flanked by ahelical regions. In contrast, these a-helices are missing from the putative Ca2+-bindingsegments of the adhesion receptor. Overall, the four repeated putative Ca2+-binding regions in the three a-subunits contain about 109 residues (FnR,), 62 of which (57%) are conserved. The four repeats are separated from each other by stretches of about 30 amino acids of lower homology. Outside of the putative Ca2+-binding region, identical positions are generally clustered into stretches of 4-1 0 residues. The carboxyl-terminal region of the large chain (positions 837-858 of FnR,) and the amino-terminal region of the small chain (positions 1-59 of FnR,) have a very low degree of similarity (8.5%). In all three proteins, an arginine residue has been assigned to the carboxyl terminus of the large chain. The amino-terminal sequences of the VnR, (Suzuki et al., 1986) and GP 1Ib small chains (Charo et al., 1986) have been determined, and the translation of the cDNAs for these two proteins places this arginine residue in the adjacent position. The amino-terminal sequence of the FnR, small chain has not been reported, but on the basis of sequence homology, we have assigned the arginine at position 858 as the carboxyl terminus of its large chain. The three adhesion receptor a-subunit proteins differ in their number of cysteines. The FnR, has 20 cysteines: 16 in the
8162 B I O C H E M I S T R Y
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FITZGERALD ET AL.
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FIGURE 5 : Alignment of the cDNA-deduced protein sequencesof the VnR (top rows) and FnR (middle rows) a-subunits and of GP IIb (bottom rows). The VnR, protein sequence starting at position 404 is taken from the published sequence (Suzuki et al., 1986). Amino acids at an identical position in all three proteins are indicated at the top (asterisks), and those at an identical position in the VnR, and GP IIb are indicated at the bottom of each set of rows (vertical bar). (A) Large chains. (B) Small chains. Cysteines for the FnR, are numbered consecutively. The four putative Ca*+-bindingdomains are boxed with solid lines, and the transmembrane segments are boxed with dashed lines. (alpha-he1 t x )
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Amino acid sequencesillustrating the putative Ca2+-binding sequences for the adhesion receptor a-subunits and the "EF hand" structure. The Ca*+-bindingcoordinatesare shown on the bottom, and the correspondingresidues are boxed. BBCalm I1 is bovine brain calmodulin, second binding repeat (Gari5py & Hodges, 1983). Secondary structure is shown at the top. Residues indicated by upper-case letters in FnR in the a-helical regions show positions not favoring a-helix, while the upper-case letters in the BBCalm sequence indicate residues favoring a-helix. large chain and 4 in the small chain. If these are numbered consecutively, then the VnR, is missing cysteines 15 and 20, while G P IIb is missing cysteines 11, 15, and 20. All three proteins have six cysteines in the amino-terminal region of the large chain, whereas the region of the putative Ca*+-binding
repeats contains no cysteines. The large and small chains of G P IIb are apparently joined by a single interchain disulfide, since incubation of platelet GP IIb-IIIa with an increasing concentration of j3-mercaptoethanol results in a separation of the large and small chains of G P IIb, followed by an increase in molecular weight of the small chain? Thus, two of the three cysteines in the G P IIb small chain are in the form of an intrachain disulfide, which leaves only a single cysteine available to form the interchain disulfide. If all of these proteins have a single interchain disulfide, then free sulfhydryls may be present in the large and small chains of FnR, and the large chain of GP IIb. DISCUSSION The presence of endothelial cell membrane proteins related to the platelet G P IIb-IIIa complex has been established independently in a number of laboratories (Fitzgerald et al.,
ADHESION RECEPTOR a - S U B U N I T S
VOL. 26, NO. 25, 1987
LARGE CHAIN 2w
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1985b; Thiagarajan et al., 1985; Leeksma et al., 1986; Newman et al., 1986; Plow et al., 1986b). Recently, we reported that the primary amino acid sequences of G P IIIa in platelets and HUVE cells are identical (Fitzgerald et al., 1987). However, cDNAs identified by using an oligonucleotide probe designed from the sequence of a platelet GP IIb peptide corresponded to that of the FnR,. Subsequently, probes based on the G P IIb cDNA sequence of HEL cells detected no positive clones. Recently, it was reported that G P IIIa and the @-subunitof the VnR are immunologically similar and have identical amino-terminal peptide sequences (Ginsberg et al., 1987). In this report, we have demonstrated the presence of VnR, clones in a HUVE cell library. Thus, it appears that the previously described endothelial cell G P IIb-IIIa like proteins are, in fact, the VnR. The terms “cytoadhesins” (Plow et al., 1986b) and “integrins” (Hynes, 1987) have been proposed to describe the adhesion receptor family, which consists of a/&subunit heterodimers. The three @-subunits, which define the three subfamilies, are of similar size (769-803 amino acids long) (Tamkun et al., 1986; Fitzgerald et al., 1987; Kishimoto et al., 1987), and they have 56 cysteines, 4 cysteine-rich tandem repeats, a carboxyl-terminal transmembrane segment, a short cytoplasmic domain, and a highly homologous region from amino acids 110 to 350. These heterodimer receptors mediate cell adhesion by interacting primarily through Arg-Gly-Asp sequences present on their ligands (Ruoslahti & Pierschbacher, 1986; Wright et al., 1987). The GP IIb-IIIa complex and the VnR can be purified by affinity chromatography using immobilized Arg-Gly-Asp-containing peptides (Pytela et al., 1986), and Arg-Gly-Asp-containing peptides will inhibit cell adhesion (Ruoslahti & Pierschbacher, 1986) and platelet aggregation (Gartner & Bennett, 1985; Haverstick et al., 1985). Leukocyte recognition of C3bi-containing particles, mediated by the Mac-l receptor, also appears to occur by an Arg-Gly-Asp-dependent process (Wright et al., 1987). The FnR has been identified in human and hamster cell lines by affinity chromatography (Pytela et al., 1985a) and by using polyclonal and monoclonal antibodies (Brown & Juliano, 1985, 1986). The mammalian FnR is often referred to as the 140-kDa complex because both the a- and &subunits have molecular weights of 140000 on reduced SDS-polyacrylamide gels. The mammalian FnR is specific for fibronectin (Pytela et al., 1986), while a homologous three-protein complex (bands 1-3) in chickens, the CSAT antigen, can bind both fibronectin (Horwitz et al., 1985; Akiyama et al., 1986) and laminin (Horwitz et al., 1985). The VnR was first described in osteosarcoma cells by affinity chromatography on either vitronectin-Sepharose or Sepharose linked to a synthetic peptide containing the Arg-Gly-Asp sequence (Pytela et al., 1985b). The GP IIb-IIIa complex is a fibrinogen, fibronectin, and von Willebrand factor receptor required for the aggregation of stimulated platelets (Plow et al., 1986a). The present study establishes the structure of FnR,, one of the a-subunits of the adhesion receptor family. Our identi-
fication of the cDNA-derived sequence as the FnR, depends on three lines of evidence. First, a published partial cDNA sequence for a FnR a-subunit (Argraves et al., 1986) is identical for 866 nucleotides with the cDNA sequence in the present report. Second, the amino-terminal sequences of the five VLA have been reported (Takada et al., 1987b). The amino-terminal sequence of the a-subunit of VLA-5 (Takada et al., 1987b), which is thought to be the FnR, matches the predicted sequence shown in Figure 3. Third, we have detected the presence of the 140-kDa FnR complex in HUVE cells by using polyclonal antib~dies.~ The three a-subunits have several structural features in common (Figure 7). The proteins consist of two disulfidelinked polypeptide chains (Argraves et al., 1986; Suzuki et al., 1986; Poncz et al., 1987), and the cleavage of the propeptide appears to occur at an Arg residue (Argraves et al., 1986; Poncz et al., 1987). All three a-subunits have a single transmembrane segment, which is located near the carboxyl terminus of the small chain. This location leads to the prediction of a short cytoplasmic domain of 20-32 residues. The extracellular large chains are linked by a single interchain disulfide bond to the small chains. The three a-subunits are homologous (Suzuki et al., 1986; Poncz et al., 1987); the alignment of their protein sequences results in overall identities of 36.1-44.5%. Identical residues appear clustered, most notably in the four repeated segments that contain sequences of amino acids characteristic of Ca2+-binding proteins (Poncz et al., 1987). The putative Ca2+-bindingsequences of the adhesion receptor a-subunit differ from those of other Ca2+-binding proteins in three respects. First, the a-helices before and after the Ca2+-binding @-turns in proteins such as calmodulin, troponin C, and parvalbumin (GariBpy & Hodges, 1983), i.e., the thumb and forefinger of the so-called EF hand (Kretsinger & Nockolds, 1973), appear to be absent from the putative Ca2+-bindingsites of the adhesion receptor a-subunits. The glycine and proline residues in these regions (Figure 6) have a low probability of forming a-helices as analyzed by the Chou and Fasman (1978) rules. Second, the -Z positions of the Ca2+-bindingsites of calmodulin, troponin C, and parvalbumin are invariably occupied by Glu, most likely because of its strong helix-forming potential. In the adhesion receptor asubunits, however, these positions are occupied by Val or Ile, which does not form strong helices. Third, the spacing between the putative Caz+-bindingsites in the a-subunits of the adhesion receptors (43-55 residues) differs from those of other Ca*+-bindingproteins (18-28 residues). The importance of Ca2+is best understood for the platelet GP IIb-IIIa complex. Chelation of Ca2+causes dissociation of GP IIb-IIIa, either in detergent solutions (Kunicki et al., 1981) or on intact platelets (Fitzgerald & Phillips, 1985). The role of Ca2+in the structure and function of other adhesion receptor complexes has not been determined; however, it is known that the VnR I. Charo, L. Bekeart, and D. R. Phillips, unpublished
observations.
8164 B I O C H E M I S T R Y of HUVE cells cannot be dissociated by Ca2+ chelation (Fitzgerald et al., 1985b; Leeksma et al., 1986). The protein sequence homologies among the a- and @subunits of the adhesion receptor family suggest that they evolved by gene duplication. Alignment of the sequences of the P-subunits showed that the chicken FnR/laminin receptor band 3 (equivalent to the human FnR, and VLA,) has a higher homology to GP IIIa and the leukocyte @-subunit (45.4%) than does GP IIIa to the leukocyte @-subunit(38.5%).2 Similarly, the FnR, has higher homology to GP IIb and the VnR, than does GP IIb to the VnR, (Table I). This indicates that the precursors to the FnR a-and @-subunitscould have given rise to the other adhesion receptor subunits. The subfamily of adhesion receptors defined by the FnR/VLA @subunit appears to be more diverse than the other two subfamilies; this subfamily has at least five separate a/@heterodimers, and its a-subunits consist of both single-chain and two-chain proteins (Hynes, 1987). A group of Drosophila proteins, termed "position-specific antigens", resembles the adhesion receptors. They have an a//?-heterodimeric structure, with subunits of M , 110 000150000 (Wilcox & Leptin, 1985; Leptin, 1986). The position-specific antigens have unique a-subunits complexed with a common @-subunit,and their role in embryogenesis may involve cell migration and adhesion. The amino-terminal sequence of one of the Drosophila a-subunits is homologous to the adhesion receptor a-subunits (Takada et al., 1987b). The cDNA-deduced protein sequence for several a- and &subunits offers the possibility of a greater understanding of their functional roles as mediators of cell adhesion and platelet aggregation. Studies to determine the locations of ligand recognition sites, the regions involved in the noncovalent association of the subunits, and the sites of possible interaction with the cytoskeleton will elucidate the relative importance of conserved and divergent regions that regulate the common and the specific functions in this receptor family. ACKNOWLEDGMENTS We thank Dr. Stuart Orkin of Harvard Medical School and Dr. David Ginsberg of The University of Michigan for providing the HUVE cell cDNA library, Drs. John Taylor, Brian McCarthy, and Beatriz Levy-Wilson for help in planning this study, Drs. Brian Blackhart and Stephen Lauer for advice on experimental procedures, Erwin Ludwig, Vince Pierotti, and Richard Otter for advice on DNA sequencing and sequence analysis, Kate Sholly and Theresa Cardinale for manuscript preparation, James X. Warger for graphics and photography, and Sally Gullatt Seehafer and A1 Averbach for editorial assistance. Registry No. Receptor, fibronectin (human clone 1-7/1-3 asubunit heavy chain protein moiety reduced), l l 1237-06-0; receptor, fibronectin (human clone 1-7/1-3 a-subunit light chain protein moiety reduced), 1 1 1237-07-1; receptor, vitronectin (human a-subunit heavy chain protein moiety reduced), 1 1 1237-08-2;glycoprotein IIb (human HEL cell heavy chain protein moiety reduced), 1 1 1237-04-8; glycoprotein IIb (human HEL cell light chain protein moiety reduced), 1 1 1237-05-9; calcium, 7440-70-2.
REFERENCES Akiyama, S. K., Yamada, S. S., & Yamada, K. M. (1986) J . Cell Biol. 102, 442-448. Argraves, W. S., Pytela, R., Suzuki, S., Millan, J. L., Pierschbacher, M. D., & Ruoslahti, E. (1986) J . Biol. Chem. 261, 12922-12924. Brown, P. J., & Juliano, R. L. (1985) Science (Washington, D.C.)228, 1448-1450.
FITZGERALD ET AL.
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Crystal Structures of Metyrapone- and Phenylimidazole-Inhibited Complexes of Cytochrome P-45OCamt Thomas L. Poulos* and Andrew J. Howard Protein Engineering Department, Genex Corporation, Gaithersburg, Maryland 20877 Received February 18, 1987; Revised Manuscript Received June 15, 1987
The crystal structures of metyrapone- and 1-, 2-, and 4-phen limidazole-inhibited complexes of cytochrome P-450,,, have been refined to a nominal resolution of 2.1 l a n d compared with the 1.63-A camphor-bound structure. With the exception of 2-phenylimidazole, each of the inhibitors forms an N-Fe bond with the heme iron atom while part of the inhibitor sits in the camphor-binding pocket. In the 2-phenylimidazole complex, a water molecule or hydroxide ion coordinates with the heme iron atom while the inhibitor binds in the camphor pocket adjacent to the aqua ligand. Each of the inhibitors forces the central region of helix I that forms part of the O2binding pocket to move away from the inhibitor, with the exception of 2-phenylimidazole where the helix moves in toward the inhibitor. In addition, the Tyr-96 region, which provides specific contact points with the substrate, is perturbed, although to varying degrees with each inhibitor. These perturbations include large, localized changes in Debye-Waller or temperature factors, indicative of changes in dynamical fluctuations. The largest inhibitor, metyrapone, causes the fewest changes, while 2-phenylimidazole binding causes the largest, especially in helix I. The large 2-phenylimidazole-induced movement of helix I can be rationalized on the basis of the inhibitor imidazole group's hydrogen-bonding requirements. ABSTRACT:
Cytochromes P-450 are a widely occurring class of b-type heme proteins that catalyze the hydroxylation of both aliphatic and aromatic molecules (Hayaishi, 1974). Much of what is known about the details of cytochrome P-450 structure and function stems from studies with cytochrome P-450,, obtained from the soil bacteria Pseudomonas putida (Wagner & Gunsalus, 1982; Gunsalus et al., 1974; Debrunner et al., 1978; Gunsalus & Sligar, 1978). P-450,, catalyzes the conversion of camphor to 5-exo-hydroxycamphor(Gelb et al., 1982) and is the first step in the oxidative assimilation of camphor when camphor is utilized by P. putida as a carbon source (Gunsalus et al., 1974). +This work was supported in part by NIH Grant GM 33688. *Address correspondence'to this author at the Center for Advanced Research in Biotechnology, 9600 Gudelsky Drive, Rockville, MD 20850.
0006-2960/87/0426-8 165$01.50/0
The crystal structure of P-450,, has been determined with substrate bound first at 2.6 8, (Poulos et al., 1985) and then 1.63 8, (Poulos et al., 1987). In addition, the substrate-free structure has been refined at 2.1 8, (Poulos et al., 1986). The substrate, camphor, contacts the heme ring immediately adjacent to the oxygen-binding site and is held in place by complementary contacts with neighboring aliphatic and aromatic residues in addition to a single hydrogen bond between the camphor carbonyl oxygen atom and the hydroxyl group of Tyr-96. When camphor is bound, the high-spin heme iron atom is pentacoordinate with the sulfur atom of Cys-357 serving as the only axial ligand (Poulos et al., 1985). In the absence of camphor, the substrate pocket fills with a hydrogen-bonded array of solvent molecules, one of which coordinates with the iron atom (Poulos et al., 1986), giving a hexacoordinate, low-spin heme. No significant conformational 0 1987 American Chemical Society
