Calcium-binding proteins.

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Copyright 1976. All rights reserved

Annu. Rev. Biochem. 1976.45:239-266. Downloaded from www.annualreviews.org by Middle Tennessee State University on 08/20/13. For personal use only.

CALCIUM-BINDING PROTEINS Robert H. Kretsinger1 Department of Biology, University of Virginia, Charlottesville, Virginia 22901

CONTENTS \.

2.

PERSPECTIVES AND SUMMARY.............................................................................. CATALOG OF CALCIUM-BINDING PROTEINS . ...... ... ... ...... ...... ...... .. .. ........ ......... ..

2.1 2.2

Regulatory Proteins of Muscle ....... .................................. ............................... Calcium-Modulated Intracellular Factors ................................................. ......

2.3

Calcium-Modulated Intracellular Enzymes......................................................

2.4 2.5

Cytoplasmic Enzymes with Possible Calcium Interaction ......................... ...... Mitochondrial Enzymes with Possible Calcium Interaction..............................

2.6 2.7 2.8

Calcium-Binding Proteins Unique to the Nervous System................................

2.9

Myosin Adenosine Triphosphatase

.

.

.

.

240 246 247 248 248 252

2.10 Other Intracellular Calcium-Binding Proteins of Eukaryotes ....... .. ...... .........

252 252 255 255

2.11 Extracellular Calcium-Binding Enzymes of Eukaryotes .. .. ............. ....... .. .. .. 2.12 Olher EXlracellular Calcium-Binding Proleins of Eukaryoles . .. ... .. . . .... ...

257 258

2.13 Extracellular Calcium-Binding Enzymes of Prokaryotes .......... ....... ... .. .......... 2.14 Calcium-Binding Glycoproteins .. ... . ..... . . . ..... . ...... . ...... . ... ... . . .... 2.15 Calcium-Binding Protein of Phage PM2 ........................................................

259 259 260

GENERALIZATIONS AND CONCLUSIONS . ..... " .... . . . . . . . " ........... "... . . . . . . . . .

261 261 262

Vitamin D-Induced Calcium-Binding Proteins................................................ Membrane Adenosine Triphosphatases.............................................................. """"''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''

.

.

..

.

3.

239 240

.

..

.

. ...

.

... .

.

..

.

... .

.

.

.

.

. . .

.

3.1 Calcium Coordination ..... ..................... "...... .................. .... ..................... ........ 3.2 Calcium as a Second Messenger-Activation and Modulation .

"""""""""''''''

1 . PERSPECTIVES AND SUMMARY

Calcium binding, which appears to be either specific or physiologiCally significant, has been reported for 70 nominally "different" proteins. First, I catalog these proteins. Only a few recent or general references can be cited. Those proteins that serve critical physiological functions or that may be regarded as chemical prototypes are discussed in more detail. Second, I present several generalizations. Inevitably the logic here is cyclic in that the generalizations dictate which data from all those available are actually pre sented. In order to focus our attention in the catalog, I outline these generalizations: IRecipient of National Institutes of Health Career Development Award GM-70480.

239


240

KRETSINGER

1. An examination of the five calcium-binding proteins of known structure does not reveal a correlation between ligand type and/or geometry and CaH affinity or selectivity. 2. For many of the enzymes supposedly activated or stabilized by CaH the available data do not allow one to judge the physiological significance of the calcium binding or its contribution to the enzymic mechanism. 3. Of the enzymes requiring Ca2 and concanavalin A, only the nuclease of Staphylococcus and possibly phospholipase appear to bind Ca2 at the active site. 4. The calcium affinities of most of the extracellular enzymes are low, pKd = 3 to 4. This is consistent with the fact that the Ca2+ concentration of the extracellular environment is about 10-3 M. 5 . The cytosol concentration of free CaH in most if not all eukaryotic cells is from 10-6 to 10-8 M. Following a stimulus to the cell, after which CaH functions as a second messenger, the free CaH may rise to 10-6 to 10-5 M. The reported affinities of most enzymes of the cytosol are too low to be physiologically signifi cant. 6. Several intracellular enzymes or enzyme activators have pKd values between 5 and 8. These enzymes therefore may be turned on and off or "modulated" in response to extracellular stimuli. 7. There is a distinct conceptual difference between extracellular enzymes that are "activated" by CaH and those intracellular enzymes that are modulated by CaH. The activated proteins bind CaH upon secretion or upon incorporation into a secretory vesicle and retain it throughout their functional lifetimes. The modu lated proteins may bind and release CaH many times in response to varying concentrations of this second messenger. 8. Several of the calcium-modulated proteins contain a characteristic conformation, consisting of a helix, calcium-binding loop, and second helix, referred to as the "EF hand." These proteins are homologous, that is, evolutionarily related. 2. CATALOG OF CALCIUM-BINDING PROTEINS 2.1

Regulatory Proteins of Muscle

Muscle calcium-binding parvalbumin (MCBP) appears to function as a mediator of CaH in the white muscle of vertebrates (I, 2); however. its specific function remains unknown. The crystal structure (3) consists of six a-helical regions. A through F. A calcium ion is bound in the loop between helices C and D. A second CaH is coordinated in the EF loop. Helix C. the CD loop. and helix D are related to the EF region (the "EF hand") by an intramolecular. approximate twofold axis (Figure I). The coordi nation of both the CD and the EF CaH can be represented by octahedra (see figure legend). This assignment illustrates four points: (a) The two octahedra are related by an intramolecular. approximate twofold axis. as are the CD and EF hands. (b) The

CALCIUM-BIN DIN G PROTEINS

241


C\

\ @\ .

( // EF

HAND

Figure 1

CD and EF regions of calcium-binding parvalbumin from carp muscle. Helix C,

the CD calcium-binding loop, and helix D of carp MCBP are related by an intramolecular approximate twofold axis to the EF region. The Ca2+ coordination octahedra (Table 2) are also related by the twofold axis. The loops are in /3-antiparallel sheet conformation and are connected by one hydrogen bond, residues 58-97. Helix D is somewhat distorted from a regular a-helix, supposedly because of its interaction with the invariant Arg-75, Glu-81 hydrogen bond. The EF region has been chosen as the evolutionary prototype, or "EF hand," as drawn to the right. The hands are viewed from the inside of the molecule.

register of ligands in the two octahedra is the same. (c) The Y ligands of both octahcdra are carbonyl oxygen atoms from Phe-57 and Lys-96. (d) Residue 98 is glycine; the -x ligand is water. The CD and EF loops form a /3-antiparallel pleated sheet connected by one main chain hydrogen bond from lie-58 to lIe-97. The EF CaH, which is coordinated by H20, is displaced by equimolar Tb3+ (4), and further addition of Tb3+ displaces the CD CaH (5). The EF CaH is removed by a 20: I molar ratio of 1,2-bis-(2-dicarboxymethylaminoethoxy)-ethane (EQ TA) but the CD CaH is not [(5), and D. J. Nelson et aI, manuscript submitted for publication]. -


242

KRETSINGER

Residues Arg-75 and Glu-8 1 are invariant and form an internal polar hydrogen bond. This bond is functionally coupled to the Ca2+-binding loops, even though both CaH ions are over 20 A away. Reaction of Arg-75, the only arginine present, with cyclohexanedione causes the loss of two antigenic determinants, a reduced a-helix content, and the loss of half of the Ca2+-binding capacity of MCBP (6l . Removal of the EF Ca2+ makes the sole -SH group, Cys- 1 8, which is only 5 A from the Arg-7S, G1u-8 1 hydrogen bond, much more reactive to S,S'-dithiobis-(2-nitroben zoic) acid (DTNB) (7). The NMR label, trifluoroacetonyl, attached to this -SH shows a large shift following removal of the EF CaH and an even larger shift after removal of both CaH (5). MCBP evolved from a "primitive" EF hand by repeated gene duplication and fusion (8). The AB region subsequently had two amino acids deleted and lost its CaH-binding ability. The CD region and the EF main chains are superimposable to 1 .0 A and are related by the intramolecular, approximate twofold axis. This EF hand is the basic evolutionary unit for the calcium-binding component of troponin and for the myosin light chains (Tables I and 2). Crayfish tail muscle contains a water-soluble protein that differs from parvalbu min and troponin. It has two subunits, each of 2 1 ,SOO mol wt, and binds Ca2+, with pKd = 6.5 and n = 4 per dimer (9). Myosin ATPases are indirectly modulated by calcium. This control is exerted either via troponin bound to the actin and tropomyosin of the thin filament or via myosin light chains associated with the myosin heavy chains of the thick filament. The troponin trimer ( 10, 1 1 ) consists of three dissimilar subunits, referred to as TNT (mol wt 37,000) , TNI (mol wt 23,000) , and TNC (mol wt 1 7,846). As illus trated in Figure 2 [adapted from ( 1 2)], in the resting or 10w-Ca2+ state tropomyosin binds to actin in a position where it blocks actin-myosin interaction. The inhibitory component of troponin (TNI) binds both tropomyosin and actin and is responsible for holding tropomyosin in the blocking position. TNT binds to tropomyosin in both states; it is at the pivot point. The calcium-binding component (TNC) undergoes a significant conformational change upon binding Ca2+. Although it does not interact directly with either actin or tropomyosin, it does bind to both TNT and TN!. Following the binding of Ca2+ by TNC, the affinity of TNI for actin is greatly reduced. Then tropomyosin shifts deeper into the grooves of the actin double helix ( 1 3) along its entire 385 A length. This movement is highly cooperative since there is only one troponin, occurring every 385 ..t, for seven actins. TNC has two sites for Mg2+(pKd = 3.5), two for Ca2+(pKd 6.7), and two that bind either CaH(pKd 8.7) or MgH(pKd = 3.5) ( 14). The affinity of TNC for Ca2+ is greatly enhanced by its interaction with TNI. There is no cooperativity in Ca2+ binding. During a relaxation-contraction-relaxation cycle the CaH concentra tion ranges from "' 10-8 to "' 1 0-5 M. The Mg2+ concentration in the sarcoplasm is in the range 10-4 to 1 0-3 M; hence the high-affinity (CaH or MgH) site would appear to be occupied by a divalent cation at all times. Based on the homologies in amino acid sequence (Table 1 ) between MCBP and TNC ( I S}, Kretsinger & Barry ( 1 6) proposed that the structure of TNC consists of four EF hands arranged in two pairs. =

=


Table 1

Amino acid sequences of homologous calcium-binding p rot ein s3 ,

MCBP AB

6

L

N

D

A

MCBP-CD

3SK SA D

MCBP E - r

77L

T

TNC I

14E M

TNC 2

soP

TNC 3 TNC4

D

I

1

AAA L EACKAA DS

1

DVKKAF A I

1

D GE T K T

r

r KA

A

D A

l

AE

TK EE

I

I

L

I

1

126 VT DEE

I

I

r

1

.1

SIN P G 13 2

AC L c

M K s IsK

T

r

I r DA D GG G

1-

1

1-

F

D GSG

A

K VG

K

L

F

E E DE

L

I

1

G V DE F

TA

I

I

L

L Q N

-I D r EE F

T

-I

D AEE

F

L

-I DE F

I

L V M A

T

I

S37

F KA D A, RA76

I

E

I

K

M

1

I I

I

L

1

.

'

L V KA 10S

-I SVKE LGTV MR M

D

D R N A DG Y

I-

D H KA F F

G Q T49

MV R Q M KE

r

1

RAS G E H

I

D AKG S9

I2S

M E G V QIS9 I I L T G D SK T L S QV G D V L RA L G Tso I I I I G , , �K, S � � N AEVKKV L N K N P DE Q M N AK 1 E r E Q r LP M L QA 1 S N

AC L iJ AC L 'Y

-

E S L M K D G DK N

AC L !>

Vit D CaBP

D M

l EE V D E

I

I I 46EQQ D E F KE AF

T

IDQ DKSG

L KAG D S D G DG K

90KSEE E L AE C F R

I

F

1

1-

b

YE

I

1

1

1

D F VEG L

E EEVEA

141 QKE

RV L

N

l LY D R

D

DKE

DG

1-

T

VG MGAE L R

M AGQE DS NGC

x

L

-I

1

I II G r F KQ L L VSV QKAG EK

Ligand

-I I

Ir

DG R

y

z

-y

HV

-I NYE A F VK H D

-X

KES

L

1

Q

D

L

1 1 1

T

1

A

I

M S

119 0

T

L

L

2 � 0,

1

I

z

KSG P so

o

-z

Z

aThe sequences are aligned to show the homologousEr hand regions. There has apparently been a deletion of two amino acids in theAB

re gion

of parvalbumin and an insertion of three in the (l-region of the alkali-extractable light chain of myosin. The underlined residues are (supposedly)

involved in calcium

coordination. The coordination octah edra

(supposed) inner aspects of the !>-helices. A ( laA,ArgR, Asn

Pro P,SerS, Thr T, Trp W, Tyr Y,ValV). bThe abbreviations used in Tables I and 2

are

N,

shown

in Figure 1 and summarized in Table

2.

Th e vertical lines indicate the

Asp D,CysC,GIn Q, GluE, GlyG, His H, lIe I ,Leu L , LysK, Met M,Ph e e ,

are as follows: MCBP, muscle calcium-binding parvalbumin from carp (AB,CD, and Er regions);

TNC, calcium-binding component of troponin from rabbit skeletal muscle;ALC, alkali-e)(tractable light chain of rabbit skeletal myosin;Vit D

CaBP, vitamin D-induced calcium-binding protein from b ovine gut.

("'J >

h

L G EIS7

L

F

D QI22

Cl "tl

';I:i

� tTl Z en N � w

244-

KRETSIN GER

Table 2

a,b A summary of crystal structures of calcium-binding proteins X


Protein

Y

Z

-Y

-X

MCBP-CO

D-51

0-53

MCBP-EF

5-55

1'-57

[-59

0-90

[D-nl

D-94

K-96

TNC I

D-n

0-29

H2O

D-33

H2O

5-35

-z [-62 [[-1011

l;

C�O

H2O

- CO2

pKd

6

0

4

>7

8

I

4

E-38

I

>7 5.5,7.5

TNC 2

0-63

D-65

$-67

T-69

D-71

[-74

0

4

5.5,7.5

TNC 3

D-I03

N-I05

D-107

Y-109

D-III

E-114

0

4

5.5,7.5

TNC 4

D-139

N-141

D-143

R-145

D-147

E-150

6

0

4

5.5,7 5

ALC"

0-59

T-61

D-63

K-65

T-67

Q-70

6

0

2

ALC),

D�136

E-138

ALC 6

(M-144)

(6)

0

0-173

H2O

V-142

Q-171

C-I77

N-I79

Vi' D CaBP Con A

[0-10'1

N-175

5- 29

0.31

A-33

0-35

Y-12

D-19'

N-14

H2O

H2O

Nuclease

D-19

D-21

Thermolysin

D-\38

E-I77*

E-177'

N-183

U-57

D-59

Y-193

T-194

T-194

E-70

N-n

H2O

2 4 Trypsin

[-147

H2O

6

5-38

(5)

>6

H2O

7

- 4

E-43

0-40

T-41

6

H2O

E-187

D-185*

[-190*

6

D-185'

E-190'

Q-61

H2O

H2O

H2O

2'H2O

E·80

H2O

1-197 V-75

H2O

I'

4

- 3

2.5

4.7

i.5 6

4.7 >6

D-200

>6

H2O

3.4

aThe Ca2+ ligands are assigned to octahedral vertices as illustrated for MCBP in Figure 1. The first residue in amino

acid sequence defines the +X vertex. the second +Y vertex. All residues coordinate with oxygen atoms. except Met-144

of ALe 1'. Residues using peptide oxygens are underlined. The two oxygen atoms of a carboxyl group may both co· ordinate one Ca2 t, indicated by brackets, and/or may coordinate two different cations,indicated by an asterisk. In thermolysin site 4, two water molecules coordinate; both are near the -Y vertex. bSee footnote b. Table I, for definition of abbreviations.

Figure 2

Model of actin, tropomyosin, troponin, and myosin interaction. The thin filament

is viewed in cross section. Actin,TNI, TNC, andTNT are represented as spheres proportional to their molecular weights. TNI and TNT can be cross-linked with tartryldiazide (12a) and hence are within 6

A of one another. Tropomyosin consists of two strands of a-helix. A pair A. Over a repeat length of 770 A there are 28 actin monomers,

of troponins occurs every 385

4 troponins, and 4 tropomyosin double strands. In the relaxed state (left), tropomyosin blocks the interaction of the myosin head with actin. Upon binding of Ca2+ toTNC, theTNI-actin contact is broken; tropomyosin shifts deeper into

the groove of the actin helix ( 1 3); and myosins can interact with actin along the entire length of the thin filament.


CALCIUM-BIN DIN G PROTEIN S

245

Muscle myosin and probably cytoplasmic myosin contain three or four light chains (mol wt 15,000 to 25,000) in addition to the two heavy chains (mol wt 2 10,000) that have the ATPase- and actin-combining activity (see section 2.9). In l110llusks and some other invertebrates there is no troponin associated with the thin filament and the tropomyosin lies deep enough in the groove of the actin helix so that it does not hinder actin-myosin interaction. Mollusk myosin contains one E DTA-extractable light chain and two -SH-containing light chains per molecule (17). Removal of the E DTA light chain completely desensitizes the myosin to Ca2+ regulation; however, the isolated E DTA light chain does not bind Ca2+. Both native and resensitized myosin bind about 1.4 moles of CaH with pKd = 6.5. In the flight muscle of the insect Lethocerus corda/anus, both thick and thin filaments bind Ca2+ ( 1 8). The muscle is synergistically controlled. In muscles where calcium regulation appears to act via the thin filament, there is also a direct binding of CaH to myosin. Huxley (19) obtained X-ray diffraction patterns of toad semiten dinosus muscles that had been stretched so far as to preclude any overlap of thick and thin filaments. Upon addition of physiological amounts of calcium there ap peared to be a movement of the myosin head away from the thick filament. He offered as one explanation that there may be "a second activation mechanism present in vertebrate striated muscle [that] holds the cross-bridges away from the actin filaments in a resting muscle and releases them during contraction." Skeletal muscle myosin binds two Ca2+ with pKd = 4. 9 . Ca2+ (pKd = 5.5) induces a reversible conformational change in myosin that is evinced by increased sedimen tation coefficient and reduced viscosity (20). Tryptophan fluorescence data for the DTNB-extractab1e light chain indicate pKd values of 5.2 and 4.0, with n = 2, for CaH binding and pKd 3.0, with n I, for Mg2+ (2 1 ). The Chelex method gave pKd (CaH) = 4.9. with n = 1 (20). When myosin is treated with DTNB. the actomyosin complex loses CaH sensitivity in parallel with the release of DTNB light chains (22). Rabbit DTNB light chain, which does bind CaH, can replace E DTA light chain, which in its isolated form does not bind Ca2+, in sensitizing mollusk myosin (23). Skeletal myosin also contains two alkali-extractable light chains (ALC) (mol wt 20,700 and 16,500) whose amino acid sequences are identical except that the heavier chain has 41 additional residues at the N terminus (24). Four regions within ALC appear to be homologous to the EF hands of MCBP and of TNC (25, 26) (Table 1). Nonetheless, the isolated ALC does not bind Ca2+. In summary, mollusk myosin contains one E DTA-extractable light chain that confers Caz+ sensitivity on myosin but does not bind Caz+, and two -SH-containing light chains. Skeletal myosin contains two alkali-extractable light chains that do not bind CaH, and two DTNB-extractable light chains with pKd (CaH) = 5.2 and =

n =

=

2.

The alkali-extractable light chains. like TNC. probably consist of two pairs of EF hands. The DTNB light chain and the E DTA light chain may also be homologs. The calcium-binding affinities of these light chains will depend not only on the ligands in their respective calcium-binding loops but also on conformational distor tions induced by their binding to the heavy chains of myosin, just as the Ca2+ binding affinity of TNC increases following binding to TN!.

246

KRETSINGER

DTNB light chain, TNT, and TNI are, or can be, phosphorylated; Ca2+ is probably not involved. Nonetheless many systems, like muscle, which are subject to rapid calcium regulation, might also be subject to a more subtle, long-term potentiation via phosphorylation (27).


2.2 Calcium-Modulated Intracellular Factors The universal role and importance of cyclic AMP (cAMP) as a second messenger is well established [see (28, 29) for reviews]. Ca2+ plays an equally broad role as a second messenger (29, 30). It is hardly surprising that cAMP should be involved in Ca2+ regulation and that Ca2+ should effect adenyl ate cyclase and phosphodies terase (30-32). Mammalian tissues have several cyclic GMP (cGMP) and cAMP phosphodies terases (PDE). The main peak of activity upon elution from DEAE-cellulose is dependent upon a calcium-binding phosphodiesterase activator protein (PAP). When assayed in the presence of excess activator protein and at millimolar levels of substrate, the Km of phosphodiesterase for cAMP decreases sevenfold while Km for cGMP decreases twentyfold. The activation sequence seems to be: PAP

+

Ca2+�PAP*Ca2+

PAP*Ca2+

+

PDE 10w affinity';:::::=:::' (PAP*Ca2+)PDE high affinity'

Spectral measurements suggest a conformational change in the activator protein upon binding Ca2+; the calcium-free form does not bind phosphodiesterase (33). In 1972, Wolff & Siegel originally described two calcium-binding proteins: CaBP-I and CaBP-II (34). CaBP-I has been found only in brains (section 2.7). CaBP-II, from pig brain, adrenal medulla, and testes, was subsequently found to function as a "calcium-dependent regulator (CDR) of brain cyclic nucleotide phos phodiesterase" (35). They found that "low concentrations of cyclic GMP are more effective than equivalent concentrations of cyclic AMP in promoting the association of the two proteins." The implied mechanism is:

CDR*Ca2+

+

PDE.cGMP �(CDR*Ca2+).(PDE*.cGMP).

The same calcium-dependent regulator also activates brain adenylate cyclase (E C 4.1.1.1) a hundred times more effectively than it activates guanylate cyclase (E C 4.6.1.2) (36). The physiological function of the regulator appears to be coactivation

of adenylate cyclase and of a phosphodiesterase with cGMP as its preferred sub strate. The possible identity of PAP and CDR (formerly CaBP-1I) is as yet unresolved, but would appear likely in spite of apparent differences in reported physical proper ties. PAP has been reported with molecular weights of 1 5 ,000 (31 ) 27,000 (33), and I, and isoelectric point (pI) 4.1. CDR 33,000 (34), with pKd (Ca2+) 5.5, n has a molecular weight of 11,500 (34) with a pKd (Ca2+) 4.7 (34) or 5.4 (37), n I, and pI 4.2. However, the various molecular weight determinations may ,

=

=

=

=

=

=


CALCIUM-BINDING PROTEINS

247

be a consequence of the different degrees of (Ca2-dependent aggregation (34), the different conditions and physical procedures employed in the determinations, and/ or the heating procedures employed in the purification of PAP (31, 33) as con trasted with CDR (34). PAP has recently been found to activate adenylate cyclase (37a). Wang et al (33) found that the amino acid compositions of PAP and the TNC are similar and suggested their homology. Adenylate cyclase activities have been reported in various membrane prepara tions. Many of these are either inhibited by the addition of Ca2+ or stimulated by E GTA, but the enzymes are not well enough characterized to test for a direct calcium interaction [e.g. from testis (38), brain (39), erythrocyte (40), and parotid gland (41»). 2.3 Calcium-Modulated Intracellular Enzymes The conversion of phosphorylase b to phosphorylase a by phosphorylase b kinase (EC 2.7.1.38) is stimulated by Ca2+. The apparent pKd (Ca2+) is 6.6 at pH 8.2 and 6.3 at pH 6.8; one ion is bound per 55,000 amu (42). The Ca2+ binding is specific and rapid. This Ca2+ modulation couples glycogenolysis to contraction in skeletal muscle. The phosphorylase b kinase of blowfly flight muscle is Ca2+-activated with apparent pKd 6.8 (43). The blowfly kinase, unlike that of rabbit, is 30% active in the presence of EDTA and is also activated by preincubation with 10-2 M phosphate. Marine coelenterates have two types of luminescent proteins (44). One type includes aequorin (mol wt 28,000), obelin (45), mnemiopsin, and berovin. These emit light, even in the absence of O 2, when exposed to 10-8 M CaH. =

Aequorin + Ca2+ � aequorin Ca2+ (with oxyluciferin) (with oxyluciferin monoanion*), •

Aequorin CaH apoaequorin (with oxyluciferin monoanion*) •

--

+

Ca2+

+

oxyluciferin

+

light (469 nm).

The second type, such as the luciferase (mol wt 38,000) from sea pansy, requires O2 . Calcium sensitivity derives from the separate luciferin binding protein (LBP), (mol wt 20,000). LBP-luciferin Luciferin Luciferase

•

+

O

2

+

+

Ca2+

luciferase

(oxyluciferin monoanion*)

[Mg2+) : Ca-ATP will not complex with the two -SH groups and is bound in the catalytic site. The physiological significance of this Ca-ATP hydrol ysis is not known. 3. Actin: during in vivo function, actin "covers" one or both -SH groups, thereby displacing bound Mg-ATP and causing the "initial burst" of ATP hydrolysis . 4. -SH blocking: if one of the -SH groups is chemically modified, the Mg-ATP cannot chelate and is obliged to bind at the active site. 2. 10 Other In tracellular Calcium-Binding Proteins of Eukaryotes These proteins have few functional, structural, or evolutionary properties common to one another or to those discussed in other sections. Actin (mol wt 4 1 ,785), along with tropomyosin and troponin (section 2. 1 and Figure 2), forms the thin filament of muscle. Probably all eukaryotic cells contain actin in the form of microfilaments. The acrosomal process of Limulus sperm is formed by the polymerization of actin (98). Guinea pig spermatozoa require 0.2 mM CaH in the external medium before undergoing the acrosome reaction and activa tion . (99). NMR results suggest that calcium functions as a bridge in binding ATP to actin via the /3- and 'Y-phosphate groups ( 100). A physiological binding of Ca2+ has not been demonstrated. CaH binding to thin filaments alters their circular dichroic (CD) spectrum and makes them more flexible ( 10 1). Microtubules are found in all eukaryotic cells either in specialized structures as cilia, neurotubules, and spindles or within the cytoplasm. They appear to have no inherent contractility. During spindle formation and dissolution the microtubules undergo polymerization and depolymerization. Inoue et al ( 102) have suggested that this may be the driving force for drawing the chromosomes to the poles. Ca2+ at 1.0 mM ( 103) both inhibits polymerization and causes depolymerization of tubulin. Since Ca2+ serves as a second messenger in many processes involving microtubuIes ,

256

KRETSINGER

in particular cell division, it is important to understand the calcium effect in vitro and its relationship to these physiological processes. The spasmoneme is a contractile organelle which causes flexure of the stalk of VorticelIids. About 60% of the organelle's protein has a molecular weight of 20,000, binds one to two Ca2+ per mole, and has pH 4.S. In the presence of 10"-6 to 1 0-8 M CaH it contracts reversibly to one fourth its resting length, This reaction does not require ATP and does not appear to involve covalent bond formation ( 1 04).


As noted in section 2.8 both calsequestrin and high-affinity CaBP are extrinsic membrane proteins on the inside of the SR. High-affinity CaBP may be involved in transferring Ca2+ from ATPase (Ca) to calsequestrin, which serves as a CaH-

binding resin. As indicated by CD measurements, calsequestrin undergoes a signifi cant conformational change upon CaH binding; high-affinity CaBP does not. In rabbit skeletal muscle there are two closely related isotypes of calsequestrin. Homozygotes for both forms have been identified (76). Concanavalin A (Con A) (105), produced by the jack bean ( Canavalia ensifor mis), agglutinates animal cells and is mitogenic for lymphocytes. The monomer has a molecular weight of 25,500 and 237 residues ( 106). From pH 4.5 to 5.6 it exists as a dimer, above p H 5.6 as a tetramer. Con A contains no a-helices ( 105, 106). Some 50% of the residues are in two antiparallel IJ-pleated sheets. Con A :1as a MnH (internal) and CaH (external) double site and, 20 A away, a nonreducing gluco- (or manno-) pyranoside-binding site. MnH is coordinated by the carboxyl groups of Glu-8, Asp-IO, and Asp-19, and by His-24 as well as by two water molecules. The two aspartate carboxyl groups also coordinate the CaH (Table 2), which is 4.6 A from MnH. The more internal S-1 site binds transition metal ions, normally MnH, and must be filled before the CaH is bound. From NMR and CD studies Barber & Carver ( 1 07) concluded that Mn2+ binding causes few conformational changes, that Ca2+ binding alters the S- I site as well as distant points, and linally that sugar binding induces further changes. The binding sequence is MnH (or other transition metal ion), then CaH with a large conformational change, and finally sugar ( 1 07-109). Sugar binding reduces the affinity (110) of an undetermined third site for cell surfaces. Several pKd (Ca2+) values have been reported: 5.7

for Mn2+.Con A, pH 5.0, 22°C by UV spectra ( I l l); 3.3 for NiH.Con A, pH 5.2 22°C by equilibrium dialysis ( 1 1 2); and 3.0 for M n2+.Con A, pH 5.3 by NMR ( l OS). Water extracts of wheat flour contain a basic protein of mol wt 1 6,600 that represents 0.2% by weight of the starting flour. It binds CaH strongly and specifi cally. The protein with Ca2+ binds "stearoyl-2 Iactylate, a dough improver" to form a birefringent precipitate ( 1 1 3). The light-harvesting pigment protein (mol wt 33,000) of spinach chloroplast, which binds one molecule of chlorophyll a and one of chlorophyll b, has two classes of Ca2+-binding sites: I (pKd = 5. 6, n = 1 ) and II (pKd 4.5, n = 4). Binding at site II induces "grana stacking" ( 1 14). =


257

Phosvitin (mol wt 40,(00) is a metal-binding storage protein found in egg yolk. Table 6 shows its binding properties for Ca1+ and Mg2+. Grizzuti & Perlmann ( 1 1 5) noted that of 255 total amino acid residues, 1 36 are phosphorylated (serine or threonine) and 3 1 are either aspartic or glutamic acid. They argue that at pH 6.5 the dianionic -OPO �- groups each bind one divalent cation, but at pH 4.5, -OP03H- binds neither Ca2+ nor Mg2+.


2 . 1 1 Extracellular Calcium-Binding Enzymes of Eukaryotes

These are all degradative enzymes and, with the exception of amylase. bind CaH rather weakly (Table 3E). The enzymes may be activated or stabilized by CaH upon secretion or incorporation into a secretory vacuole. Amylases are found in a remarkable variety of plant and animal tissues as well as in bacteria (2. 1 2). It is not known whether they are homologs. Fischer & Stein ( 1 1 6) described the a-amylases as "the first true calcium metalloenzymes that have been described." Unfortunately, the Ca1+ specificity, affinity, and function have yet to be determined. Ca2+ must bind to Crotalus phospholipase A2 before dibutyryllecithin binds. Subsequent model studies on the octylamine-catalyzed methanolysis of phos phatidylcholine suggested that Ca2+ may be involved in the actual catalytic mecha nism by facilitating the transfer of a proton from water to an amine group having a pK of 7.6 ( 1 7). Bovine �-trypsin contains one Ca2+-binding site (Table 2), which was located during the crystal structure refinement ( l i S). In contrast, Abbott et al ( 1 1 9) inter pret their NMR results in terms of Gd3+ being bound by Ser- 1 90 and Asp-I 94. The cleavage of the N-terminal hexapeptide (Val.Asp.Asp.Asp.Asp.Lys) of trypsinogen, which weakly binds a second Ca2+, proceeds l OO-fold faster in the presence of about 1 mM Ca2+. At least three of the serine proteases found in a commercial Pronase preparation appear to be homologs of trypsin and are expected to have similar Ca2+-binding characteristics ( 1 20). All of these enzymes are stabilized against ther mal and chemical denaturation as well as enzymatic degradation by Ca2+. Voor douw & Roche ( 1 2 1 ) presented a model of the autolysis of thermomycolase in which there are two very similar conformers, with and without Ca2+; only the Ca2+-free conformer is degraded. Free Ca2+, about 1 .0 mM as normally found in plasma, is required for the stability and activity of at least four serine proproteases-factors X (Stuart), IX (Christmas), VII, al1d II (prothrombin)-and a transglutaminase in the cascading sequence of Tahle 6

Binding affinities of phosvitin for Ca

2+

and Mg

2+

Ion

pH

/l

Ca2+ Mg 2 +

6.5

160

6 .5

160

Ca

4.5

32

4.5

40

2+

Mg2+

;; ( 1 0-

3

M)

pKd 4.1 4.1


258

KRETSINGER

enzymes whose ultimate goal is the production of a fibrin clot. Further, Ca2+ interacts directly with fibrin (2. 1 2). At least three of the factors (X, IX, and II) are homologous ( 1 22). These three and factor VII are all glycoproteins (cf 2. 14) contain ing hexoses, N-acetylhexosamine, and N-acetylneuraminic acid; 25% of the weight of factor IX is glycan. After synthesis, several glutamic acid groups of all four factors are y-carboxylated in a vitamin K-dependent reaction ( 1 23, 1 24). At least in the case of prothrombin there is one Ca2+ bound per y-carboxyglutamic acid residue. The y-carboxylglutamic acid residues are all near the N terminus and are in the fragment cleaved off during activation. They appear to function in binding the proenzyme to a phospholipid micelle at the time of activation. Fibrin-stabilizing factor (XIII) is activated to fibrinoligase, a transglutaminase that cross-links fibrin:

Ca2+ (25 mM) is required for 50% unmasking of the essential thiol ( 1 26). Price ( 1 30) has summarized the recent work on pancreatic DNase I, and has shown that at 2.5 mM Mg2+ and no Ca2+ the enzyme is inactive. With trace amounts of Ca2+ it cleaves only one strand; acting on dI.dC it cleaves the dI strand, gen'erat ing 5'-phosphate termini. Optimal activity is obtained at }O-4 M Ca2+. Ca2+ binds to the enzyme without substrate and is required for substrate binding. 2. 1 2 Other Extracellular Calcium-Binding Proteins 0/ Eukaryotes The elastic modules of fibrin clots in 8.}O-3 M Ca2+ is 1 50 dyn/cm2 while the modulus is 75 dyn/cm2 for clots in calcium-free medium ( 1 3 1). Elastin occurs in high concentration in arteries and has been suggested as a Ca2+ nucleation site in plaque formation. Elastin contains some 30% glycine. Urry proposed a model of a super helix consisting of a sequence of ,8-bends. He suggested that several peptide carbonyl oxygen atoms would form a neutral site that could coordinate Ca2+ ( 1 32). If elastin is solubilized by alkali, the binding capacity is one Ca2+ per 2.5 X IQ4 amu. Abatangelo et al ( 1 33) hydrolyzed elastin with 0.25 M oxalic acid, thereby solubilizing 60% of the fiber. At pH 8 one Ca2+ is bound per 7 X 103 amu with an apparent pKd of 4.5. At pH 5 only half as much Ca2+ is bound but with the same affinity. Because of the pH dependence of the binding, they argue that most of the binding involves carboxylate groups and is probably nonspecific. Up to 4.3 mM the Ca2+ binding by casein is eng6thermic; at higher concentration, micelles are formed and the process is exothermic ( 1 34). It is not known whether the eight phosphoserine residues of aSI -casein (mol wt 23,6 1 6) are involved in calcium coordination. Hemocyanins are nonheme, copper glycoproteins found free in the hemolymph of Mollusca and Arthropoda where they transport O ( 1 35, review). The mollusk 2 protein has at least six aggregation forms. The largest is a cylinder 280 A in diameter and 360 A long, of 9,000,000 mol wt. The monomer appears to have a mol wt of


259

225,000 and binds eight Cu and four 0 ' Disaggregation is favored by high pH and 2 low CaH. For Levantina hierosolima pKd (CaH) 1 . 9 with n 80 per "-'200,000 amu ( 1 36); for Dolabella auricularia pKd 3.6, n = 1 2 3 ( 1 37). The activity of human pancreatic lipase (EC 3. 1 . 1 .3) is stimulated sixfold by the addition of an activator found in pancreatic juice whose interaction requires 0.8 mM CaH and 2.0 mM deoxycholate. The activator (mol wt 1 3 ,(00) is 9% hexose, 1 7 % hexosamine, and 2% sialic acid ( 1 38). =

=

=


2. 1 3 Extracellular Calcium-Binding Enzymes of Prokaryotes Bacteria secrete a variety of degradative enzymes that bind Ca2+ (Table 3F). For several of the amylases and proteases Ca2+ is required for optimal activity as well as thermostability. In the crystal structure of Staphylococcus nuclease ( 1 39) the substrate analogue thymidine 3',5'-diphosphate is apparently bound to Ca2+ by a bridging water mole cule (Table 2, and section 3. 1 ). CaH is required for both activity and proper folding ( 1 40). TJiermolysin has a double site for Ca2+ that is 14 A away from the Zn2+ at the active site ( 1 4 1). The two calcium ions at the double site are 3.8 A apart and lie in a cleft between the two lobes of the molecule. Their coordination and those of the surface sites 3 and 4 are summarized in Table 2 and discussed in section 3. 1 . All four calcium ions protect against autolysis, whereas only one, at site 3 or 4, contributes toward thermostability ( 1 42). Neutral protease A, a homolog of thermo lysin, lacks sites 3 and 4 and is denatured at 6Q°C ( 1 43), whereas thermolysin is stable at 85°C. Like thermolysin its Hill coefficient for binding at the internal double site is 2.0. Contrary to what one might expect, the surface sites with only two (site 3) and one (site 4) carboxyl groups have pKd > 6, whereas the double site with a total of four carboxyl groups has pKd = 4.7.

2. 1 4 Calcium-Binding Glycoproteins Several glycoproteins of known enzymic function (clotting factors X, IX, VII and II-section 2. 1 1 ), hemocyanin, and lipase-activating factor (section 2 . 1 2) have al ready been mentioned. The sponge aggregation factor is involved in the specific adhesion of sponge cells and serves as an important model for factors controlling both malignant and normal tissue development. Dissociated sponge cells from Microciona prolifera and Hali clona occulata aggregate in a species-specific manner if Ca2+ is present in the seawater. The sponge aggregation factor from Microciona parthena is a "sunburst" shaped proteoglycan of mol wt 2. 1 X 107 that is comprised of an inner circle of mol wt 5 X 106 and about 14 radiating glycoprotein arms about 1000 A long. It consists of 49% glycan, of which 2 1 % is uronic acid, and 47% protein, of which 28% are aspartic and glutamic acid. There are 1 1 50 calcium-binding sites per 2 . 1 X 107 amu 3.5. If the Ca2+ concentration is lowered, the arms irreversibly with a pKd dissociate into subunits of mol wt 2 X 105; if it is raised, the factor aggregates into a gel. It is not known how the factor attaches to the cell surface, or how it confers its specificity ( 1 47). =

260

KRETSINGER

Goblet cell mucin is one of the major components of mucus and may significantly affect the amount of calcium accessible for intestinal absorption. In cystic fibrosis the calcium content of mucus increases, the glycoprotein becomes more compact, and the excess mucus produced forms a "mucus plug." The molecule has a molecu lar weight of 2 X 106, 88% glycan, 34 disulfide bonds, and an axial ratio of 225: I ( 148).


A glycoprotein from human saliva forms an aggregate of molecular weight 5.5 X 106 in the presence of 10-2 M Ca2+. It is assumed that its calcium-binding capacity is accounted for by its 10% saccharide content and its fi v e phosphate groups per 1 1 ,000 mol wt subunit ( 1 49). The glycoprotein (mol wt 200,000) from pre-osseous cartilage has two classes of

Ca2+-binding sites: pKd = 7, n = i; and pKd 4, n = 600 . It may be involved in bone formation ( 1 50). According to Gomez-Puyou et al ( 1 5 1), "intact rat liver mitochondria contain respiration-independent high affinity CaH binding sites whose properties suggest =

they may be concerned in respiration-dependent CaH transport." These site.s ac count for 0.5 to 1 .0% of the protein of mitochondria, and contain 27% phospholipid and 8% hexosamine. Sottocasa et al ( 1 52) have described two high-calcium-affinity

glycoproteins from vertebrate mitochondria. The first has a molecular weight of 42,000, of which 30% is phospholipid and 5% carbohydrate. It is water-soluble, whereas

the rat Iipoglycoprotein is not. The second has a molecular weight of

30,000, of which 1 5 % is carbohydrate.

Vitellogenin is an estrogen-induced calcium-binding protein found in the serum of the toad, Xenopus laevis. It contains 1 2% phospholipid and I % carbohydrate and runs as a single band on 4% acrylamide gel electrophoresis ( 1 53). Vitellogenic proteins are specific to adult females and are ultimately sequestered into protein yolk bodies. They are also found in insects but there is no suggestion that the insect vitellogenin binds calcium. A low molecular weight glycopeptide, which binds 20 equivalents of CaH, has been isolated from fungal cell wall matrix ( 1 54). It may be involved in CaH trans port or facilitated diffusion.

2. 1 5

Calcium-Binding Protein of Phage PM2

The icosohedral bacteriophage PM2 contaihs a DNA core, an inner capsid, a lipid layer extending radially from 200 to 240 A, and an outer capside from 240 to 300 A. The lipid bilayer contains 64% of the acidic phosphatidylglycerol (some 440 molecules), most of which is on the outer surface. This capsid contains 820 copies of protein II (mol wt 26,000) whose pI is 1 2.3. Schafer et al ( 1 55) measured the pI of protein II as 9.0 following guanidine denaturation, which they found necessary to release CaH. In contrast Snipes et al (1 56) state that "no significant amount of calcium is bound stably to the virion." They note that the host, Pseudomonas BAL-3 1 , a marine bacterium, grows in 3 X IG-4 M Ca2+, b u t requires 3 X 1 0-5 M for phage production. They suggest that CaH is bound to protein II only transitorily prior to its generally assumed interaction with phosphatidylglycerol.


26 1

3. GENERALIZATIONS AND CONCLUSIONS 3 . 1 Calcium Coordination


The approximately octahedral coordination geometries ofthe five proteins of known structure and of the predicted structure of troponin subunit C (TNC) are summa rized in Table 2. The following observations are pertinent. I . The coordination number is usually six. The carboxyl groups of Asp-92 and Glu- l O l in MCBP and Asp- l O in Con A coordinate with both oxygen atoms. Hence the formal coordination numbers are eight and seven. In order to maintain the formal octahedral presentation, two water molecules are assigned to the y vertex of the seven-coordinate site 4 of thermolysin. 2. No nitrogen or sulfur ligands have been observed. 3. The number of water ligands ranges from zero to three. 4. In all of the complexes a main-chain carbonyl oxygen atom is involved; three are at site 4 of thermolysin. 5. Two of the carboxyl groups in Con A bridge calcium and manganous ions separated by 5.3 A. In thermolysin Ca- l and Ca-2 are 3.8 A apart, bridged by three carboxyl groups. 6. The protein results are consistent with the precedents derived from the structures of20 organocalcium complexes ( 1 ). The coordination number in small complexes is usually eight or less frequently seven. 7. Contrary to common assumption there is not a correlation between the number of carboxyl ligands and calcium affinity. Nonetheless most of the proteins (MCBP, TNC, phosphodiesterase activator protein, calcium-dependent regula tor vitamin D CaBP, S- lOO, CaBP-I, L- l , and a-amylase) that bind Ca2+ strongly and specifically are acidic. -

The crystal structures of these proteins as well as a general consideration of the others do not reveal the basic rules for Ca2+ specificity, particularly relative to Mg2+. The Ca2+ of many of these proteins-MCBP, thermolysin, a-amylase, phos pholipase, Con A, trypsinogen-can be displaced by lanthanides ( I , 1 57). In general the affinity for the lanthanide is greater than that for Ca2+; the degree of isomor phism varies greatly. In thermolysin one lanthanide displaces both Ca2+ of the double site; at sites 3 and 4 the lanthanide is within 0.4 A of the Ca2+ position ( 1 58). Several of these proteins are phosphorylated: casein, phosvitin, and the glyco proteins from saliva and from mitochondria. There is no indication that the Ca2+ actually coordinates to phosphate oxygens. In contrast Ca2+ appears to be coordi nated by the carboxyl groups of y-carboxylglutamate residues of the clotting factors ( 125). In Staphylococcus nuclease the Ca2+ is near the active site, but the catalytic mechanism is not yet understood. Wells ( 1 1 7) suggested that Ca2+ is involved in hydrolysis by phospholipase A2 (section 2. 1 1). For the other enzymes there is little evidence that Ca2+ is at the active site, even though, as in the case in DNase,

262

KRETSINGER

CaH binding precedes substrate binding. For most, CaH is bound at a site removed from the active site. Thermostability and resistance to proteolysis is imparted to nearly all the ex tracellular enzymes and factors. With Ca2+, thermolysin is stable up to 85°C. Boiling is used in the preparation of lipase-activating factor and of phosphodieste rase activator protein.


3.2 Calcium as a Second Messenger-Activation and Modulation Most of the extracellular enzymes are not only stabilized but are also partially activated by 10-3 to l Q--4 M CaH as is normally found in the extracellular environ ment. With free Ca2+ concentrations in the r;nge of 10-7 M in the cytosol, these degradative enzymes are not activated prior to their secretion or incorporation into a secretory granule. Exposure to relatively high extracellular concentrations of Ca2+ (as well as zymogen cleavage) appears to be one of the mechanisms involved in activating these enzymes. The a-amylases from both eukaryotes and prokaryotes do not fit this simple scheme, since they are active in 10-7 M CaH. Usually organisms do not vary their extracellular Ca2+ concentration; however, it has been suggested that Ca2+ release from platelets might speed clotting ( 1 25) and that pancreatic secretion of Ca2+ might help digestive enzymes (159). In contrast, Ca2+-modulated factors (sections 2. 1 and 2.2) and enzymes (section 2.3) are found in the cytoplasm of eukaryotes. Their pKiCaH) values are in the range 5 to 8. They are assumed to be cyclically turned on and olfby Ca2+ functioning as a second messenger. The intracellular enzymes of section 2.4 appear to bind Ca2+ too weakly to fall within this modulation range. The relationship of the calcium concentration within the mitochondrion and calcium in the cytosol is still not well established. The functional modulation (65) of some of the mitochondrial enzymes (section 2.5) is probable but not proven. The free Ca2+ concentration within the nucleus and within prokaryotes is unknown; hence one cannot judge the physiological significance of Ca2+ binding by endonuclease or by pyridine nucleotide transhydrogenase (section 3.3). The myosin light chains, TNC, and MCBP are homologous and contain four or three EF hands (Figure I). I suggested (160) that most calcium-modulated proteins are evolutionarily related, i.e. contain EF hands, and conversely, that any protein that contains an EF hand is calcium-modulated. Further, the existence of an EF hand should be recognizable from an appropriate test of the amino acid sequence. There are certainly exceptions to this generalization, for instance the spasmonene protein (section 2 . 1 0). At least, the hypothesis has the virtue of focusing research on several key questions: What are the amino acid sequences of the intracellular enzymes of section 2.4, of the mitochondrial enzymes (section 2. 5), of the protein of the nervous system (section 2.4), and of the vitamin D-induced proteins (section 2.7)? 2. What are the precise values of the intracellular free CaH concentration and, even more challenging, the Ca2+ concentration within mitochondria, nuclei, and bac teria? Although the determination of pKd(Ca2+) may seem mundane, results 1.



from different laboratories frequently differ by factors of 1 0 to nation is as important as it is difficult.

1 00.

263

This determi

The unique physiological role of calcium was first recognized by Ringer (161) and further explored by Heilbrunn ( 162) at a phenomenological level. Douglas (163) and his colleagues found that exocytosis is usually coupled to a flux of Ca2+. Rasmussen and his colleagues (29, 30) developed the concept that calcium functions as a second messenger, often in parallel with cAMP. Analytical research on the chemical and biochemical aspects of calcium-protein interactions is proceeding rapidly. I hope this review contributes to a molecular interpretation of the seemingly diverse biological roles of calcium.

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Zinc proteins: enzymes, storage proteins, transcription factors, and replication proteins.

RNA-binding proteins: masking proteins revealed.

G proteins.

Pioneer proteins.

Folding proteins.

Geranylgeranylated proteins.

Cytotoxic proteins.

G proteins.

Characteristics of antisweet substances, sweet proteins, and sweetness-inducing proteins.

Proteins of Yaba monkey tumor virus I. Structural proteins.

Comparison of canavanyl-envelope proteins to normal envelope proteins.

Two-dimensional electrophoresis of membrane proteins: separation of myelin proteins.

RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins.

Determination of saposin proteins (sphingolipid activator proteins) in human tissues.

Thiophosphorylated proteins in chromaffin cells are chromaffin vesicle matrix proteins.

Viruses and viral proteins.

SnapShot: FMRP interacting proteins.

Glycans of myelin proteins.

New placental proteins.

Proteins of iron metabolism.

Bacterial iron-sulfur proteins.

Nervous system specific proteins.

Factor H-related proteins.

Novel immunoglobulin binding proteins.