Homology in protein sequences expressed by correlation coefficients

Internal homologies in an amino acid sequence of a protein and in amino acid sequences of two different proteins are examined, using correlation coefficients calculated from the sequences when residues are replaced by various quantitative properties of the amino acids such as hydrophobicity. To improve the signal-noise ratio the average correlation coefficient is used to detect homology because the correlation depends on the property considered. In this way, any sequence repetition in a protein and the extent of the similarity and difference among proteins can be estimated quantitatively. The procedure was applied first to the sequences of proteins which have been assumed on other grounds to contain some internal sequence repetitions, α-tropomyosin from rabbit skeletal muscle, calmodulin from bovine brain, troponin C from skeletal and cardiac muscle, and then to the sequences of calcium binding proteins, calmodulin, troponin C, and L2 light chain of myosin. The results show that α-tropomyosin has a markedly periodic sequence at intervals of multiples of seven residues throughout the whole sequence, and calmodulin and skeletal troponin C contain two homologous sequences, the homology of troponin C being weaker than that of calmodulin. Candidates for the calcium binding regions of both troponin C, calmodulin, and L2 light chain are the homologous parts having a high average correlation coefficient (about 0·5) with respect to the sequences of the CD and EF hand regions of carp parvalbumin. The procedure may be a useful method for searching for homologous segments in amino acid sequences.     

The Amino Acid Sequence of the Tryptophan-Containing Subunit (α-Subunit) of Bovine Brain S-100 Protein

Abstract: The tryptophan-containing subunit (α-subunit) of bovine brain S-100 protein was purified from a S -aminoethyl derivative of S-100a protein, and its amino acid sequence was determined. The α-subunit contained 93 residues, including one tryptophan, and had a molecular weight of 10,400. The sequence shows an extensive homology (58% identity) to the sequence of another "tryptophan-free" subunit (β-subunit) found in both S-100a and S-100b protein, and has a calcium binding site characteristic of the "E-F hand" proteins, such as calmodulin or troponin C. The tryptophan residue is located at position 90 which is presumably adjacent to the C-terminal end of the α-helix following the calcium binding loop, and thus appears likely to serve as a specific probe in structure-function studies of S-100a protein.     

Calmodulin binding to the intestinal brush-border membrane: comparison to other calcium-binding proteins

The intestinal brush-border membrane contains a high concentration of calmodulin bound to a 105,000 dalton (105 kDa) protein. Binding of radioiodinated calmodulin to this protein does not require calcium but is inhibited by trifluoperazine and excess unlabelled calmodulin. Recent evidence suggests that the 105 kDa protein in conjunction with calmodulin may be involved in the regulation of calcium transport across the brush-border membrane. In this report, we evaluated the binding of the 105 kDa protein to other radioiodinated calcium-binding proteins including the vitamin D-dependent intestinal calcium-binding protein. We observed that troponin C and S100 beta protein both bound strongly to the 105 kDa protein. The binding of S100 beta was inhibited by EGTA, but was little affected by trifluoperazine and excess unlabelled S100 beta, whereas that of troponin C was inhibited by trifluoperazine and excess unlabelled troponin C, but was little affected by EGTA. Both troponin C and S100 beta bound to a large number of proteins to which calmodulin did not bind. The vitamin D-dependent calcium-binding protein (calbindin) from chick intestine and rat kidney also bound to the 105 kDa protein, albeit more weakly than troponin C, S100 beta and calmodulin. The binding of the calbindins was increased by EGTA and was little affected by trifluoperazine and excess unlabelled calbindin. Parvalbumin, rat osteocalcin, and alpha-lactalbumin showed little binding to any brush-border membrane protein. Our results indicate that the 105 kDa calmodulin-binding protein of the intestinal brush border can bind to a variety of calcium-binding proteins all of which contain homologous regions thought to be the calcium-binding sites. Only the binding of troponin C resembles the binding of calmodulin, however, in being inhibited by trifluoperazine and excess unlabelled ligand. The functional significance of these observations in terms of regulating calcium transport across the brush-border membrane remains to be established.     

Calcium binding proteins. Elucidating the contributions to calcium affinity from an analysis of species variants and peptide fragments

This paper describes the sequence homology of calcium-binding proteins belonging to the troponin C superfamily. Specifically, this similarity has been examined for 276 twelve-residue calcium-binding loops. It has been found that, in the calcium-binding loop, several residues appear invariant, regardless of the species of origin or the affinity of the protein. These residues are Asp at position 1 (+X of the coordinating position of the calcium), Asp or Asn at position 3 (+Y), Gly at position 6, Ile at position 8, and Glu at position 12 (-Z). It has also been found that conservation of certain residues can vary in similar sites in similar proteins. For example, position 3 (+Y) in site 3 of troponin C is always an Asn, whereas in calmodulin the residue is always Asp. This study also examined the calcium-binding affinities of peptide fragments comprising the loop, helix-loop, loop-helix, and helix-loop-helix. These were compared with larger enzymatic or chemically generated protein fragments in an effort to understand the various contributions to the calcium-binding affinity of a single-site versus a two-site domain as found in troponin C and calmodulin. Based on free energy differences, it was found that a 34-residue helix-loop-helix peptide represents about 60% of the binding affinity found in the intact protein. Cooperativity with a second calcium binding site accounted for the remaining 40% of the affinity.     

A synthetic 33-residue analogue of bovine brain calmodulin calcium binding site III: synthesis, purification, and calcium binding

The sequential solid-phase synthesis of a peptide analogue of bovine brain calmodulin calcium binding site III covering residues 81-113 of the natural sequence is described. Methionine-109 is replaced by a leucine residue to avoid complications in the synthesis and purification. In an attempt to relate the structure of the calcium binding sites in the naturally occurring calcium binding protein to the calcium affinity of these sites, the synthetic analogue is examined for calcium binding by circular dichroism spectroscopy. The calcium binding characteristics are compared to those of a synthetic analogue of the homologous calcium binding site III in rabbit skeletal troponin C. The Kd of the calmodulin site III fragment for Ca2+ is determined as 878 microM whereas the Kd of the troponin C fragment is 30 times smaller at 28 microM. Structural changes induced in the peptides by Ca2+ and trifluoroethanol are similar. This study supports our contention that the single synthetic calcium binding site is a reasonable model for the study of the structure-activity relationships of the calcium binding sites in calcium-regulated proteins such as calmodulin and troponin C.     

Protein-induced inactivation and phosphorylation of rabbit muscle phosphofructokinase.

Several previously untested proteins promote the reversible inactivation of rabbit skeletal muscle phosphofructokinase. Grouped in decreasing order of effectiveness, they include the following: skeletal muscle troponin C greater than troponin, the two smooth muscle myosin light chains, alpha-actinin, and S-100 much greater than parvalbumin and soybean trypsin inhibitor. The efficiency of troponin C in this process may even exceed that previously reported for calmodulin. Sequences near calcium binding site III are apparently involved in the troponin C-phosphofructokinase interaction. Troponin C and calmodulin exert calcium-dependent effects on the physical and chemical properties of muscle phosphofructokinase. When calcium is present, comigration with either protein allows the enzyme to enter the stacking gel during urea-polyacrylamide gel electrophoresis. Both enhance the phosphorylation of phosphofructokinase catalyzed by the cAMP-dependent protein kinase, with phosphate incorporations approaching 2 mol of P/mol of protomer. Reaction occurs at Ser774 and at Ser376--a novel site whose phosphorylation is highly sensitive to troponin C and less so to calmodulin. Maximum phosphorylation has slight effect on the catalytic activity of the enzyme under standard assay conditions. The troponin C induced or calmodulin-induced phosphorylation of phosphofructokinase requires calcium and is strongly inhibited by either fructose 2,6-bisphosphate or fructose 1,6-bisphosphate. Inactivation occurs in the presence or absence of calcium, with generally higher concentrations of effectors required for protection in the latter case. Liver and yeast phosphofructokinases shows little activity loss in the presence of either calmodulin or troponin C. We have developed and tested a general mathematical model for the protein-induced inactivation of phosphofructokinase which may find application to other systems.     

Ion binding to calmodulin

Summary Over the past few years calcium has emerged as an important bioregulator. Upon external stimulation, the cell generates a transient Ca2+ increase, which is transformed into a cellular event through a molecular cascade. The first step in this cascade is the binding of calcium to proteins present in the cytosol. These proteins capable of binding Ca²⁺ under physiological conditions all belong to the same evolutionary family that evolved from a common ancestor. However, they strongly differ in the properties of their calcium binding sites. Calmodulin, the ubiquitous calcium binding protein present in all eukaryotic cells, is very close to the ancestor protein, presents four calcium binding sites which bind calcium, magnesium and monovalent ions competitively and is involved in the triggering of cellular processes. Parvalbumin, another member of the family, is more specialized and found mostly in fast-twitch skeletal muscle. It binds calcium and magnesium with high affinity and seems to be involved in muscle relaxation. On the other hand, troponin C which confers Ca²⁺ sensitivity to acto-myosin interaction exhibits both triggering and relaxing sites. The study of intracellular Ca^2– binding proteins has shown that calcium binding proteins have evolved from a simple common structure to fulfill different functions.Abbreviations CaBP calcium-binding protein - ICaBP the vitamin D-dependent intestinal Cat+binding protein - S-100 the glial S-100 protein - RLC the phosphorylatable myosin regulatory light chain - CaM calmodulin - Pa parvalbumin - TnC troponin C - TnI troponin I - Hepes N-2-hydroxyethylpipezarine, N-2-ethane-sulfonic acid - W7 N-(6-Aminohexyl)-5-chloro-l-Naphtalene sulfonamide - SDS sodium dodecyl sulfate - NMR nuclear magnetic resonance     

Isolation and characterization of calmodulin from an insulin-secreting tumour.

A major protein constituent of a rat islet cell tumour that exhibited Ca2+-dependent changes in electrophoretic mobility has been purified to homogeneity and compared in its physicochemical and biological properties with bovine brain and rat brain calmodulin (synonymous with phosphodiesterase activator protein, calcium-dependent regulator, troponin C-like protein and modulator protein). The protein, like these calmodulins, contained trimethyl-lysine, exhibited a blocked N-terminus and had an identical amino-acid composition and molecular weight on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Peptide "maps' prepared after digestion of the three proteins with trypsin, papain or Staphylococcus V-8 proteinase were virtually superimposable. Ca2+ altered the electrophoretic mobilities the enhanced the native protein fluorescence in an equivalent manner with all three proteins. Equilibrium dialysis experiments demonstrated in each case the binding of 4g-atoms of calcium/mol of protein; the binding sites were equivalent and showed Kd 0.8 microM. Tumour and brain proteins were equipotent as Ca2+-dependent activators of partially purified rat brain cyclic nucleotide phosphodiesterase, and in this action were inhibited in an identical manner by trifluoperazine. The proteins also exhibited the common property of Ca2+-dependent binding to troponin I, histone H2B and myelin basic protein. The estimated tumour content of calmodulin was 450 mg/kg fresh wt., a value similar to that reported in islets of Langerhans. These results further document the validity of the islet cell tumour as an experimental model of Ca2+-mediated molecular events associated with insulin secretion. They also suggest that brain calmodulin may be substituted for endogenous calmodulin in experimental investigations into the mechanism of insulin secretion.     

Interaction of a fluorescent N-dansylaziridine derivative of troponin I with calmodulin in the absence and presence of calcium

Rabbit skeletal muscle troponin I was covalently labeled with N-dansylaziridine, resulting in a fluorescent labeled protein. This derivative (DANZTnI) and native troponin I (TnI) inhibited calmodulin (CaM) stimulation of bovine heart Ca2+-sensitive cyclic nucleodite phosphodiesterase with identical inhibition constants. Association of DANZTnI with calmodulin was monitored directly by changes in flourescence intensity in the presence of Ca2+ and by changes in fluorescence anisotropy in the absence of Ca2+. Quantitation of the affinity of calmodulin for calmodulin-binding proteins in both the presence and absence of Ca2+ is necessary for prediction of the extent of interaction of both Ca2+ and calmodulin-binding proteins with calmodulin in vivo. The dissociation constants for the DANZTnI-calmodulin-l4Ca2+ and DANZTnI-calmodulin complexes were 20 nM and 70 micrometers, respectively. These dissociation constants define a free energy coupling of-4.84 kcal/mol of troponin I for binding of Ca2+ and troponin I to calmodulin. The Ca2+ dependence for troponin I-calmodulin complex formation predicted from these experimentally determined parameters was closely approximated by the Ca2+ dependence for complex formation between troponin I and fluorescent 5-[[[(iodoacetyl)amino]ethyl]-amino]-1-napthalenesulfonic acid derivatized calmodulin as determined by fluorescence anisotropy. Complex formation occurred over a relatively narrow range of Ca2+ concentration, indicative of positive heterotropic cooperativity for Ca2+ and troponin I binding to calmodulin.     

A model for human cardiac troponin C and for modulation of its Ca2+ affinity by drugs

Calcium sensitizers are drugs which increase force development in striated muscle by sensitizing myofilaments to Ca2+. This can happen by increasing Ca2+ affinity of the regulatory domain of Ca2+ binding protein troponin C. High resolution crystal structures of two calcium binding proteins, calmodulin (Babu et al.: J. Mol. Biol. 203:191-204, 1988) and skeletal troponin C (Satyshur et al.: J. Biol. Chem. 263:1628-1647, 1988; Herzber et al.: J. Mol. Biol. 203:761-779, 1988), have recently been published. This makes it possible to model in detail the calcium-sensitizing action of drugs on troponin C. In this study a model of human cardiac troponin C in three-calcium state has been constructed. When calcium is bound to calcium site II of cardiac troponin C an open conformation of the protein results, which has a hydrophobic pocket surrounded by a few polar side chains. Complexation of three drugs, trifluoperazine, bepridil, and pimobendan, to the hydrophobic pocket is studied using energy minimization techniques. Two different binding modes are found, which differ in the location of a strong electrostatic interaction. In analogy with the crystal structure of skeletal troponin C it is hypothezed that in cardiac troponin C an interaction occurs between Gln-50 and Asp-88, which has a long-range effect on calcium binding. The binding modes of drugs, where a strong interaction with Asp-88 exists, can effectively prevent the interaction between Asp-88 and Gln-50 in the protein, and are proposed to be responsible for the calcium-sensitizing properties of the studied drugs.     

Synthesis of a troponin C cDNA and expression of wild-type and mutant proteins in Escherichia coli

An avian fast striated muscle troponin C cDNA was designed and synthesized from six oligonucleotides using the overlap-fill in method and overproduced in Escherichia coli for the purpose of developing recombinant DNA approaches to study structure-function relationships in this calcium-binding regulatory protein. The recombinant protein isolated from E. coli functions as a bona fide troponin C in all properties that were assayed: calcium binding, calcium-dependent conformational change, calcium-dependent interaction with troponin I, and formation of a functional ternary complex with troponin I and troponin T that can confer calcium sensitivity on the actomyosin MgATPase. The initiating methionine was removed by E. coli leaving alanine as the first amino acid, as in the muscle troponin C. The first amino acid was not acetylated, but this difference from the muscle protein has no apparent effect on the function. The presence of Glu at position 99, as in turkey, versus Ala in chicken resulted in no detectable difference in comparing recombinant with chicken troponin C. A mutant in which residues 91-93 (Lys-Gly-Lys) in the D/E helical linker were deleted differs in function from wild-type troponin C in the conformational change that takes place upon calcium binding and its interaction with troponin I. Also, the mutant troponin C is impaired in its ability to form a functional complex with troponin I and troponin T that will confer calcium sensitivity on the actomyosin MgATPase.     

Conservation of residue interactions in a family of Ca-binding proteins

In the TNC family of Ca-binding proteins (calmodulin, parvalbumin, intestinal calcium binding protein and troponin C) approximately 70 well-conserved amino acid sequences and six crystal structures are known. We find a clear correlation between residue contacts in the structures and residue conservation in the sequences: residues with strong sidechain-sidechain contacts in the three-dimenesional structure tend to be the more conserved in the sequence. This is one way to quantify the intuitive notion of the importance of sidechain interactions for maintaining protein three-dimensional structure in evolution and may usefully be taken into account in planning point mutations in protein engineering.     

Structure of the platelet membrane glycoprotein IIb. Homology to the alpha subunits of the vitronectin and fibronectin membrane receptors

The platelet membrane glycoprotein IIb X IIIa heterodimer complex (GPIIb X IIIa) is the platelet receptor for adhesive proteins, containing binding sites for fibrinogen, von Willebrand factor, and fibronectin on activated platelets. GPIIb X IIIa also appears to be a member of a family of membrane adhesive protein receptors that plays a major role in cell-cell and cell-matrix interactions. GPIb is the larger component of this platelet receptor and is composed of two disulfide-linked subunits. In this report we describe the analysis of cDNA clones for human GPIIb that were isolated from a lambda gt11 expression library prepared using RNA from HEL cells. A total of 3.3 kilobases of cDNA was sequence, revealing a continuous open reading frame encoding both GPIIb subunits. The cDNA encodes 1039 amino acids: 137 constituting the smaller subunit, 871 constituting the larger subunit, and 30 constituting an NH2-terminal signal peptide. No homology was found between the larger and smaller subunits. The smaller subunit contains a 26-residue hydrophobic sequence near its COOH terminus that represents a potential transmembrane domain. Four stretches of 12 amino acids present in the larger subunit are homologous to the calcium binding sites of calmodulin and troponin C. Northern blot analysis using HEL cell RNA indicated that the mature mRNA coding for GPIIb is 4.1 kilobases in size. A comparison of the GPIIb coding region with available cDNA sequences of the alpha-chains of the vitronectin and fibronectin receptors revealed 41% DNA homology and 74% and 63% amino acid homology, respectively. Our data establish the amino acid sequence for the human platelet glycoprotein IIb and provide additional evidence for the existence of a family of cellular adhesion protein receptors.     

Localization of hydrophobic sites in calmodulin and skeletal muscle troponin C studied using tryptic fragments: a simple method of their preparation

The exposure of hydrophobic sites on calmodulin, skeletal muscle troponin C and their tryptic fragments was investigated using Phenyl-Sepharose chromatography. A strong binding of both proteins and their fragments corresponding to the NH2-terminal halves of polypeptide chain of respective proteins in the presence of calcium ions was observed. Only a weak interaction with Phenyl-Sepharose or its lack was observed under these conditions for fragments corresponding to the COOH-terminal halves of calmodulin and troponin C, respectively. The elution of the samples from Phenyl-Sepharose column with ethylene glycol gradient allowed to compare relative hydrophobicity of both proteins and their fragments. The results show that hydrophobic properties of calmodulin and troponin C are virtually preserved in their fragments obtained as a result of their cleavage by trypsin in half. They also indicated that the exposure of hydrophobic residues caused by the binding of calcium ions takes place mainly in the NH2-terminal halves of polypeptide chains of both proteins. A simple method of purification of tryptic fragments of both proteins based on the difference in the strength of their interactions with Phenyl-Sepharose is described.     

Phosphorylation of troponin I by protein kinase C: Mechanism of inhibition by calmodulin and troponin C

The mechanism by which calmodulin and troponin C influence phosphorylation of troponin I (TnI) by protein kinase C was investigated. The phosphorylation of TnI by protein kinase C requires the presence of acidic phospholipid, calcium and diacylglycerol. Light scattering intensity and fluorescence intensity experiments showed that TnI associated with the phospholipid membranes and caused extensive aggregation. In the presence of Ca²⁺, TnI-phospholipid interactions were prevented by approximately stoichiometric amounts of either troponin C or calmodulin. Troponin C was shown to completely inhibit phosphorylation of TnI by either protein kianse C or by phosphorylase b kinase. In contrast, calmodulin completely inhibited phosphorylation of TnI by protein kinase C, but had only little effect on TnI phosphorylation by phosphorylase b kinase. Inhibition by calmodulin did not appear to be due to interaction with PKC, since calmodulin mildly increased protein kinase C phosphorylation of histone III-S. The ratio of phosphoserine to phosphothreonine in protein kinase C-phosphorylated TnI remained approximately constant for reactions inhibited by up to 90% by clamodulin. TnI interactions with phospholipid and phosphorylation of TnI by PKC were also prevented by high salt concentrations. However, salt concentrations adequate to inhibit phosphorylation were sufficient to dissociate only TnI, but not protein kinase C from the membrane. These results suggest that the binding of TnI to phospholipid is required for phosphorylation by protein kinase C and that prevention of this binding by any means completely inhibited phosphorylation of TnI by protein kinase C.     

Studies of ligand binding to arrestin

A striking homology is observed between the regions 70-83 and 361-374 of the sequence of bovine arrestin and the calcium-binding loops of calmodulin and troponin C. However, the predicted alpha-helices flanking the calcium-binding site in calmodulin and troponin C are not present in arrestin. Direct measurements therefore were made in order to assess whether arrestin can bind calcium. We found that arrestin does not bind Ca2+ at physiological ionic strength, as determined by equilibrium dialysis, gel filtration, and fluorescence spectroscopy. Rapid and quantitative precipitation of arrestin occurs with Tb3+. The precipitation is reversed by EDTA and blocked by Mg2+ but not by Ca2+. Prompted by several reports, we also investigated whether nucleotides bind to arrestin. Neither ATP nor GTP binds under the conditions tested. Binding of arrestin to photolyzed, phosphorylated rhodopsin also does not influence the binding of calcium or nucleotides.