Robert H. Kretsinger

STRUCTURE AND EVOLUTION OF CALCIUM-MODULATED PROTEINS*

This review suggests that the intracellular functions of calcium are best understood in terms of calciums functioning as a second messenger. Further, when functioning as a second messenger, calcium completes its mission not by transferring charge nor by binding to lipid but by binding to specific targets, calcium-modulated proteins. This concept is broadly interpreted to include proteins involved in calcium transport. There is strong evidence that many, if not all, of these calcium-modulated proteins are homologs. Their structures and properties are contrasted to those of extracellular calcium-binding proteins which are not homologous to one another or to the intracellular calcium-modulated proteins. Finally, this line of thought leads to a suggestion of the evolutionary reason for the choice of calcium as the sole inorganic second messenger.

Evolution of EF-hand calcium-modulated proteins. I. Relationships based on amino acid sequences

SummaryThe relationships among 153 EF-hand (calcium-modulated) proteins of known amino acid sequence were determined using the method of maximum parsimony. These proteins can be ordered into 12 distinct subfamilies-calmodulin, troponin C, essential light chain of myosin, regulatory light chain, sarcoplasmic calcium binding protein, calpain, aequorin,Strongylocentrotus purpuratus ectodermal protein, calbindin 28 kd, parvalbumin, α-actinin, and S100/intestinal calcium-binding protein. Eight individual proteins-calcineurin B fromBos, troponin C fromAstacus, calcium vector protein fromBranchiostoma, caltractin fromChlamydomonas, cell-division-cycle 31 gene product fromSaccharomyces, 10-kd calcium-binding protein fromTetrahymena, LPS1 eight-domain protein fromLytechinus, and calcium-binding protein fromStreptomyces—are tentatively identified as unique; that is, each may be the sole representative of another subfamily. We present dendrograms showing the relationships among the subfamilies and uniques as well as dendrograms showing relationships within each subfamily.The EF-hand proteins have been characterized from a broad range of organismal sources, and they have an enormous range of function. This is reflected in the complexity of the dendrograms. At this time we urge caution in assigning a simple scheme of gene duplications to account for the evolution of the 600 EF-hand domains of known sequence.

The EF-hand family of calcium-modulated proteins

The EF-hand homolog proteins bind calcium (Ca2+) with dissociation constants in the micromolar range and are modulated by stimulus-induced increases in cytosolic free Ca2+. We have grouped over 160 different EF-hand homolog proteins into ten subfamilies and ten unique categories. Except for troponin-C, all subfamilies and unique EF-hand homologs represented in vertebrates can be found in the CNS. In this review, structural and functional characteristics of these proteins are discussed, with special emphasis on the multifunctional regulatory protein, calmodulin. The possible function of bending within the central helix of calmodulin is considered and is illustrated with a model calmodulin--target complex.

Classification and evolution of EF-hand proteins.

Forty-five distinct subfamilies of EF-hand proteins have been identified. They contain from two to eight EF-hands that are recognizable by amino acid sequence as being statistically similar to other EF-hand domains. All proteins within one subfamily are congruent to one another, i.e. the dendrogram computed from one of the EF-hand domains is similar, within statistical error, to the dendrogram computed from another(s) domain. Thirteen subfamilies - including Calmodulin, Troponin C, Essential light chain, Regulatory light chain - referred to collectively as CTER, are congruent with one another. They appear to have evolved from a single ur-domain by two cycles of gene duplication and fusion. The subfamilies of CTER subsequently evolved by gene duplications and speciations. The remaining 32 subfamilies do not show such general patterns of congruence; however, some - such as S100, intestinal calcium binding protein (calbindin 9kd), and trichohylin - do not form congruent clusters of subfamilies. Nearly all of the domains 1, 3, 5, and 7 are most similar to other ODD domains. Correspondingly the EVEN numbered domains of all 45 subfamilies most closely resemble EVEN domains of other subfamilies. Many sequence and chem-ical characteristics do not show systemic trends by subfamily or species of host organisms; such homoplasy is widespread. Eighteen of the subfamilies are heterochimeric; in addition to multiple EF-hands they contain domains of other evolutionary origins.© Kluwer Academic Publishers

Interaction of calcium and calmodulin in the presence of sodium dodecyl sulfate

Calmodulin has been purified to homogeneity using an improved procedure that allows rapid processing of several kilograms of bovine brain. A calcium-dependent change in the electrophoretic mobility of calmodulin in the presence of sodium dodecyl sulfate (SDS) has been observed. Freshly prepared calmodulin or lyophilized calmodulin, stored at --80 degrees C for 1--7 months, migrates as a single band with an apparent molecular weight of 21 000 when the sample, gel and running buffer are made 0.1 mM in EDTA. When 0.1 mM CaCl2 is substituted for EDTA, freshly isolated calmodulin migrates as a single band with an apparent molecular weight of 15 000. More slowly migrating bands, in addition to the 15 000 molecular weight band, are observed when the stored protein is electrophoresed under the same conditions. Calcium binding experiments show that freshly prepared calmodulin binds 4 mol of calcium per mol of protein in the presence of 0.1% SDS in 0.1 mM CaCl2. Skeletal muscle troponin C, carp parvalbumin, and bovine brain S-100b do not show this mobility change. The calcium-dependent mobility change can be used to identify calmodulin in crude protein preparations. Calmodulin has been identified in the sperm of the sea urchin, Strongylocentrotus purpuratus, and purified. The urchin calmodulin activates cyclic nucleotide phosphodiesterase to the same extent as does brain calmodulin. We used several criteria to determine that calmodulin is not present as a soluble protein in Escherichia coli.

Evolution of EF-hand calcium-modulated proteins. II. Domains of several subfamilies have diverse evolutionary histories.

SummaryIn the first report in this series we described the relationships and evolution of 152 individual proteins of the EF-hand subfamilies. Here we add 66 additional proteins and define eight (CDC, TPNV, CLNB, LPS, DGK, 1 F8, VIS, TCBP) new subfamilies and seven (CAL, SQUD, CDPK, EFH5, TPP, LAV, CRGP) new unique proteins, which we assume represent new subfamilies.The main focus of this study is the classification of individual EF-hand domains. Five subfamilies—calmodulin, troponin C, essential light chain, regulatory light chain, CDC31/caltractin-and three uniques—call, squidulin, and calcium-dependent protein kinase-are congruent in that all evolved from a common four-domain precursor. In contrast calpain and sarcoplasmic calcium-binding protein (SARC) each evolved from its own one-domain precursor. The remaining 19 subfamilies and uniques appear to have evolved by translocation and splicing of genes encoding the EF-hand domains that were precursors to the congruent eight and to calpain and to SARC.The rates of evolution of the EF-hand domains are slower following formation of the subfamilies and establishment of their functions. Subfamilies are not readily classified by patterns of calcium coordination, interdomain linker stability, and glycine and proline distribution. There are many homoplasies indicating that similar variants of the EF-hand evolved by independent pathways.

Crystal structure of calmodulin

The crystal structure of calmodulin has been determined to 3.6 A resolution. At this resolution the polypeptide chain can be traced. Some of the side chains have tentatively been identified. Refinement of the structure with x-ray diffraction data measured to 1.65 A resolution is continuing. As reported by Babu et al. calmodulin is about 65 A long and 30 A in diameter. Homolog domains 1 and 2 are related by a local twofold axis, as in parvalbumin and in troponin C, and form one end of the molecule. Domains 3 and 4 form the other end. The second alpha-helix of domain 2 and a short interdomain region are continuous with the first helix of domain 3, thereby forming a single helix from residues 67-93. The central region, residues 75-84, of this long helix forms a handle connecting the two pairs of homolog domains. Exclusive of the residues, 75-84, in the handle the closet approach of side chains of pair 1, 2 to pair 3, 4 is 12 A. The spatial relationship of pair 1, 2 to pair 3, 4 is similar in calmodulin to the relationship of the corresponding pairs in troponin C. However, in troponin C there are three additional residues in the handle region of the long alpha-helix and the two pairs are about 5.0 A further apart. On the surface of pair 1, 2 in calmodulin there is one extended region with many hydrophobic side chains from both domain 1 and domain 2. This hydrophobic patch is bounded by two distinct clusters of anionic side chains, one from the beginning of the first helix of domain 1 and on the other side of the hydrophobic surface one from the beginning of the first helix of domain 2. Homologously, the hydrophobic patch on the surface of pair 3, 4 is bounded by two clusters of aspartate and glutamate residues. Either or both of these hydrophobic surfaces may be sites to which calmodulin target proteins bind.

Crystal structure of phenylalanine-regulated 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli.

BACKGROUND In microorganisms and plants the first step in the common pathway leading to the biosynthesis of aromatic compounds is the stereospecific condensation of phosphoenolpyruvate (PEP) and D-erythrose-4-phosphate (E4P) giving rise to 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP). This reaction is catalyzed by DAHP synthase (DAHPS), a metal-activated enzyme, which in microorganisms is the target for negative-feedback regulation by pathway intermediates or by end products. In Escherichia coli there are three DAHPS isoforms, each specifically inhibited by one of the three aromatic amino acids. RESULTS The crystal structure of the phenylalanine-regulated form of DAHPS complexed with PEP and Pb2+ (DAHPS(Phe)-PEP-Pb) was determined by multiple wavelength anomalous dispersion phasing utilizing the anomalous scattering of Pb2+. The tetramer consists of two tight dimers. The monomers of the tight dimer are coupled by extensive interactions including a pair of three-stranded, intersubunit beta sheets. The monomer (350 residues) is a (beta/alpha)8 barrel with several additional beta strands and alpha helices. The PEP and Pb2+ are at the C-ends of the beta strands of the barrel, as is SO4(2-), inferred to occupy the position of the phosphate of E4P. Mutations that reduce feedback inhibition cluster about a cavity near the twofold axis of the tight dimer and are centered approximately 15 A from the active site, indicating the location of a separate regulatory site. CONCLUSIONS The crystal structure of DAHPS(Phe)-PEP-Pb reveals the active site of this key enzyme of aromatic biosynthesis and indicates the probable site of inhibitor binding. This is the first reported structure of a DAHPS; the structure of its two paralogs and of a variety of orthologs should now be readily determined by molecular replacement.

Super-motifs and evolution of tandem leucine-rich repeats within the small proteoglycans--biglycan, decorin, lumican, fibromodulin, PRELP, keratocan, osteoadherin, epiphycan, and osteoglycin.

Leucine‐rich repeats (LRRs) with 20–30 amino acids in unit length are present in many proteins from prokaryotes to eukaryotes. The LRR‐containing proteins include a family of nine small proteoglycans, forming three distinct subfamilies: class I contains biglycan/PG‐I and decorin/PG‐II; class II: lumican, fibromodulin, PRELP, keratocan, and osteoadherin; and class III: epiphycan/PG‐Lb and osteoglycin or osteoinductive factor. Comparative sequence analysis of the 34 available protein sequences reveals that these proteoglycans have two types of LRRs, which we call S and T. The type S LRR is 21 residues long and has the consensus sequence of xxaPzxLPxxLxxLxLxxNxI. The type T LRR has 26 residues; its consensus sequence is zzxxaxxxxFxxaxxLxxLxLxxNxL. In both “x” indicates variable residue; “z” is frequently a gap; “a” is Val, Leu, or Ile; and I is Ile or Leu. These type S and T LRRs are ordered into two super‐motifs—STT with about 73 residues in classes I and II and ST with about 47 residues in class III. The 12 LRRs in the small proteoglycans of I and II are best represented as (STT)4; the seven LRRs of class III as (ST)T(ST)2. Our analyses indicate that classes I/II and III evolved along different paths after the establishment of the precursor ST, and classes I and II also diverged after the establishment of the precursor (STT)4. Proteins 2000;38:210–225.

Structural analysis of leucine-rich-repeat variants in proteins associated with human diseases

Abstract.A number of human diseases have been shown to be associated with mutation in the genes encoding leucine-rich-repeat (LRR)-containing proteins. They include 16 different LRR proteins. Mutations of these proteins are associated with 19 human diseases. The mutations occur frequently within the LRR domains as well as their neighboring domains, including cysteine clusters. Here, based on the sequence analysis of the LRR domains and the known structure of LRR proteins, we describe some features of different sequence variants and discuss their adverse effects. The mutations in the cysteine clusters, which preclude the formation of sulfide bridges or lead to a wrong paring of cysteines in extracellular proteins or extracellular domains, occur with high frequency. In contrast, missense mutations at some specific positions in LRRs are very rare or are not observed at all.