Phosphohydrolase and transphosphatidylation reactions of two Streptomyces phospholipase D enzymes: covalent versus noncovalent catalysis

A kinetic comparison of the hydrolase and transferase activities of two bacterial phospholipase D (PLD) enzymes with little sequence homology provides insights into mechanistic differences and also the more general role of Ca(2+) in modulating PLD reactions. Although the two PLDs exhibit similar substrate specificity (phosphatidylcholine preferred), sensitivity to substrate aggregation or Ca(2+), and pH optima are quite distinct. Streptomyces sp. PMF PLD, a member of the PLD superfamily, generates both hydrolase and transferase products in parallel, consistent with a mechanism that proceeds through a covalent phosphatidylhistidyl intermediate where the rate-limiting step is formation of the covalent intermediate. For Streptomyces chromofuscus PLD, the two reactions exhibit different pH profiles, a result consistent with a mechanism likely to involve direct attack of water or an alcohol on the phosphorus. Ca(2+), not required for monomer or micelle hydrolysis, can activate both PLDs for hydrolysis of PC unilamellar vesicles. In the case of Streptomyces sp. PMF PLD, Ca(2+) relieves product inhibition by interactions with the phosphatidic acid (PA). A similar rate enhancement could occur with other HxKx(4)D-motif PLDs as well. For S. chromofuscus PLD, Ca(2+) is absolutely critical for binding of the enzyme to PC vesicles and for PA activation. That the Ca(2+)-PA activation involves a discreet site on the protein is suggested by the observation that the identity of the C-terminal residue in S. chromofuscus PLD can modulate the extent of product activation.     

Inhibition of Streptomyces chromofuscus phospholipase D activity by dichloro-(2,2':6',2''-terpyridine)-platinum (II) dihydrate

To determine the catalytic site of Streptomyces chromofuscus phospholipase D (PLD), which lacks an HKD motif, we examined the effects of inhibitors on the hydrolytic activity of the PLD by comparing it with cabbage and Streptomyces PLDs, which have two HKD motifs. We showed that dichloro-(2,2':6',2'-terpyridine)-platinum (II) dihydrate, a His- and Cys-directed chemical modifier, had inhibitory effects on the activities of all types of PLD examined. On the other hand, N-ethylmaleimide, a thiol-directed modifier had no such effects on PLD activity. These results suggest that the His residue plays an important role in the activity of Streptomyces chromofuscus PLD.     

Inhibition of Streptomyces chromofuscus Phospholipase D Activity by Dichloro-(2,2′:6′,2′-terpyridine)-platinum (II) Dihydrate

To determine the catalytic site of Streptomyces chromofuscus phospholipase D (PLD), which lacks an HKD motif, we examined the effects of inhibitors on the hydrolytic activity of the PLD by comparing it with cabbage and Streptomyces PLDs, which have two HKD motifs. We showed that dichloro-(2,2′:6′,2"-terpyridine)-platinum (II) dihydrate, a His- and Cys-directed chemical modifier, had inhibitory effects on the activities of all types of PLD examined. On the other hand, N -ethylmaleimide, a thiol-directed modifier had no such effects on PLD activity. These results suggest that the His residue plays an important role in the activity of Streptomyces chromofuscus PLD.     

Using O-(n-alkyl)-N-(N,N'-dimethylethyl)phosphoramidates to investigate the role of Ca2+ and interfacial binding in a bacterial phospholipase D

O-(n-alkyl)-N-(N,N'-dimethylethyl)phosphoramidates (n=6, 8, and 10; CnPNC) were synthesized and characterized as inhibitors of phospholipase D (PLD) activity toward phosphatidylcholine presented as monomers, micelles, and bilayers. Detailed studies with recombinant Streptomyces chromofuscus PLD, a Ca(2+)-activated enzyme that does not show large changes in catalytic activity toward the same substrate as a monomer or micelle, showed that the longer the inhibitor chain length, the more potent CnPNC is as a competitive inhibitor toward all the substrates. However, the physical state of the inhibitor did affect the maximum inhibition attainable. For a fixed concentration of diC4PC (monomer substrate), CnPNC inhibition reached a maximum around the CMC of the inhibitor; the inhibition was reduced at higher inhibitor concentrations, in part caused by the lower solubility of the aggregated inhibitor. With diC4PC as the substrate and using concentrations of C10PNC that were below its CMC, the Ki for C10PNC was 0.030+/-0.003 mM, approximately 13-fold less than the Km for substrate. Aggregated substrates showed significant inhibition of PLD by CnPNC, although as the substrate chain length increased, inhibition by a given CnPNC was diminished. With POPC vesicles, the apparent Ki for C10PNC was 0.030 of the apparent Km. The availability of these inhibitors allowed us to show that PC analogues can bind to the active site of S. chromofuscus PLD in the absence of Ca2+. Once bound at the active site, the inhibitor does not significantly affect the divalent ion-dependent partitioning of the enzyme to PC surfaces. Of the two other PLD enzymes examined, cabbage PLD, but not Streptomyces sp. PMF, was able to catalyze the cleavage of the P-N bond. Differential susceptibility of PLDs to these phosphoramidates may eventually be useful in studying PLD isozymes in cells.     

Phospholipase D in hormonal and stress signaling

Phospholipase D (PLD) is a family of diverse enzymes that are differentially regulated by Ca(2+), polyphosphoinositides, free fatty acids, G-proteins, N-acylethanolamines, and membrane lipid environments. Two new types of PLDs were identified in the past year: one is activated by oleic acid and the other requires no cation for activity. The oleate-stimulated PLD is associated with the plasma membrane and binds to microtubules. The Ca(2+)-independent PLD contains a PX and a PH domain, but not the Ca(2+)/phospholipid-binding C2 domain found in most plant PLDs. The mechanism by which Ca(2+), phosphoinositides, and G proteins regulate certain PLDs is better understood. PLDs and their product phosphatidic acid are involved in various stress responses, including water deficits, salts, wounding, and elicitation. Increasing evidence supports a role of PLD in the abscisic acid signaling cascades.     

Two highly homologous phospholipase D isoenzymes from Papaver somniferum L. with different transphosphatidylation potential

The genes of two phospholipase D (PLD) isoenzymes, PLD1 and PLD2, from poppy seedlings (2829 and 2828 bp) were completely sequenced. The two genes have 96.9% identity in the encoding region and can be assigned to the alpha-type of plant PLDs. The corresponding amino acid sequences do not contain any signal sequences. One Asn-glycosylation site, six and two phosphorylation sites for protein kinase C and tyrosine kinase, respectively, and two phosphatidylinositol-4,5-bisphosphate binding motifs could be identified. Like in most plant PLDs, two HKD motifs and one C2 domain are present. PLD1 and PLD2 have ten and nine cysteine residues. The two enzymes were expressed in E. coli and purified to homogeneity by Ca2+ ion-mediated hydrophobic interaction chromatography. The Ca2+ ion concentration needed for carrier binding of the two enzymes in chromatography as well as for optimum activity was found to be considerably higher (>100 mM) than with other alpha-type plant PLDs. Although PLD1 and PLD2 differ in eleven amino acids only, they showed remarkable differences in their transphosphatidylation activity. Two amino acid exchanges within and near the first HKD motif contribute to this difference as shown by the A349E/E352Q-variant of PLD2.     

Transphosphatidylation activity of Streptomyces chromofuscus phospholipase D in biomimetic membranes.

The phospholipase D (PLD) from Streptomyces chromofuscus belongs to the superfamily of PLDs. All the enzymes included in this superfamily are able to catalyze both hydrolysis and transphosphatidylation activities. However, S. chromofuscus PLD is calcium dependent and is often described as an enzyme with weak transphosphatidylation activity. S. chromofuscus PLD-catalyzed hydrolysis of phospholipids in aqueous medium leads to the formation of phosphatidic acid. Previous studies have shown that phosphatidic acid-calcium complexes are activators for the hydrolysis activity of this bacterial PLD. In this work, we investigated the influence of diacylglycerols (naturally occurring alcohols) as candidates for the transphosphatidylation reaction. Our results indicate that the transphosphatidylation reaction may occur using diacylglycerols as a substrate and that the phosphatidylalcohol produced can be directly hydrolyzed by PLD. We also focused on the surface pressure dependency of PLD-catalyzed hydrolysis of phospholipids. These experiments provided new information about PLD activity at a water-lipid interface. Our findings showed that classical phospholipid hydrolysis is influenced by surface pressure. In contrast, phosphatidylalcohol hydrolysis was found to be independent of surface pressure. This latter result was thought to be related to headgroup hydrophobicity. This work also highlights the physiological significance of phosphatidylalcohol production for bacterial infection of eukaryotic cells.     

Study on thermostability of phospholipase D from Streptomyces sp

Four phospholipases D (PLDs) in the culture supernatants from Streptomyces strains were purified to conduct a comparative study of their thermostabilities. Among the four purified PLDs, the enzyme from Streptomyces halstedii K1 lost its activity at 45 degrees C. PLD from Streptomyces septatus TH-2 was stable at the same temperature. We determined the nucleotide sequence encoding the PLD gene from S. halstedii K1 (K1PLD). The deduced amino acid sequence showed high homology to that of the PLD gene from S. septatus TH-2 (TH-2PLD). By comparison of the optimum temperature and the thermostability among recombinant PLDs, K1PLD, TH-2PLD and T/KPLD that possessed the N-terminus of TH-2PLD and the C-terminus of K1PLD, T/KPLD showed the properties midway between those of K1PLD and TH-2PLD. It was suggested that the 176 amino acids at C-terminus of Streptomyces PLD were important for its thermostability.     

Phospholipase D mechanism using Streptomyces PLD

Phospholipase D (PLD) plays various roles in important biological processes and physiological functions, including cell signaling. Streptomyces PLDs show significant sequence similarity and belong to the PLD superfamily containing two catalytic HKD motifs. These PLDs have conserved catalytic regions and are among the smallest PLD enzymes. Therefore, Streptomyces PLDs are thought to be suitable models for studying the reaction mechanism among PLDs from other sources. Furthermore, Streptomyces PLDs present advantages related to their broad substrate specificity and ease of enzyme preparation. Moreover, the tertiary structure of PLD has been elucidated only for PLD from Streptomyces sp. PMF. This article presents a review of recently reported studies of the mechanism of the catalytic reaction, substrate recognition, substrate specificity and stability of Streptomyces PLD using various protein engineering methods and surface plasmon resonance analysis.     

Location of the catalytic nucleophile of phospholipase D of Streptomyces antibioticus in the C-terminal half domain.

Phospholipase D (PLD) of Streptomyces antibioticus was labelled with fluorescent-labelled substrate, 1-hexanoyl-2-{6-[(7-nitro-2-1, 3-benzoxadiazol-4-yl)-amino]hexanoyl}-sn-glycero-3-phosphocholine, when it was incubated with the substrate and the reaction followed by SDS/PAGE. Mutant enzymes lacking the catalytic activity were not labelled under the same conditions, indicating that labelling of the PLD occurred as the result of its catalytic action. This confirmed that the labelled protein was the phosphatidyl PLD intermediate. PLDs contain two copies of the highly conserved catalytic HxKxxxxD (HKD) motif. Therefore, two protein fragments were separately prepared with recombinant strains of Escherichia coli. One of the fragments was the N-terminal half of the intact PLD containing one HKD motif, and the other was the C-terminal half with the other motif. An active enzyme was reconstructed from these two fragments, and therefore designated fragmentary PLD (fPLD). When fPLD was subjected to the labelling experiment, only the C-terminal half was labelled. Therefore, it was concluded that the catalytic nucleophile that bound directly to the phosphatidyl group of the substrate was located on the C-terminal half of PLD, and that the N-terminal half did not contain such a nucleophile.     

C-terminal loop of Streptomyces phospholipase D has multiple functional roles

We have recently shown that two flexible loops of Streptomyces phospholipase D (PLD) affect the catalytic reaction of the enzyme by a comparative study of chimeric PLDs. Gly188 and Asp191 of PLD from Streptomyces septatus TH-2 (TH-2PLD) were identified as the key amino acid residues involved in the recognition of phospholipids. In the present study, we further investigated the relationship between a C-terminal loop of TH-2PLD and PLD activities to elucidate the reaction mechanism and the recognition of the substrate. By analyzing chimeras and mutants in terms of hydrolytic and transphosphatidylation activities, Ala426 and Lys438 of TH-2PLD were identified as the residues associated with the activities. We found that Gly188 and Asp191 recognized substrate forms, whereas residues Ala426 and Lys438 enhanced transphosphatidylation and hydrolysis activities regardless of the substrate form. By substituting Ala426 and Lys438 with Phe and His, respectively, the mutant showed not only higher activities but also higher thermostability and tolerance against organic solvents. Furthermore, the mutant also improved the selectivity of the transphosphatidylation activity. The residues Ala426 and Lys438 were located in the C-terminal flexible loop of Streptomyces PLD separate from the highly conserved catalytic HxKxxxxD motifs. We demonstrated that this C-terminal loop, which formed the entrance of the active well, has multiple functional roles in Streptomyces PLD.     

Modulation of phospholipase D activity in vitro

Most phospholipases D (PLDs) occurring in microorganisms, plants and animals belong to a superfamily which is characterized by several conserved regions of amino acid sequence including the two HKD motifs necessary for catalytic activity. Most eukaryotic PLDs possess additional regulatory structures such as the Phox and Pleckstrin homology domains in mammalian PLDs and the C2 domain in most plant PLDs. Owing to recombinant expression techniques, an increasing number of PLDs from different organisms has been obtained in purified form, allowing the investigation of specific and unspecific interactions of the enzymes with regulatory components in vitro. The present paper gives an overview on different factors which can modulate PLD activity and compares their influence on the enzymes from different sources. While no biological regulator can be recognized for extracellular bacterial PLDs, the most prominent specific activator of eukaryotic PLDs is phosphatidylinositol-4,5-bisphosphate (PIP₂). In a sophisticated interplay PIP₂ seems to cooperate with several regulatory proteins in mammalian PLDs, whereas in plant PLDs it mainly acts in concert with Ca²⁺ ions. Moreover, curvature, charges and heterogeneities of membrane surfaces are assessed as unspecific modulators. A possible physiological role of the transphosphatidylation reaction catalyzed by PLDs in competition with phospholipid hydrolysis is discussed.     

Analysis of cell viability using time-dependent increase in fluorescence intensity

To determine phospholipase D (PLD) activity, an infrared spectroscopy assay was developed, based on the phosphate vibrational mode of phospholipids such as dimyristoylphophatidylcholine (DMPC), lysophosphatidylglycerol (lysoPG), dipalmitoylphosphatidylethanolamine (DPPE), and lysophosphatidylserine (lysoPS). The phosphate bands served to monitor the hydrolysis rates of phospholipids with PLD. The measurements could be performed within less than 20min with 10μl of buffer containing 2 to 40mM DMPC and 10 to 200ng of Streptomyces chromofuscus PLD (corresponding to 350-7000pmol of DMPC hydrolyzed per minute). The limit of sensitivity was approximately 10ng of PLD at 100mM Tris-HCl (pH 8.0) with 10mM Ca(2+) and 2.5mgml(-1) Triton X-100. Reproducible specific activity of PLD (35±5nmol of hydrolyzed DMPCmin(-1)μg(-1) PLD) measured by the infrared assay remained stable over 50 to 200ng of PLD and over 5 to 40mM DMPC. The feasibility of this assay to determine the hydrolysis rate of other phospholipids such as lysoPG, DPPE, and lysoPS was confirmed. The IC(50) of cobalt (800±200μM), a known S. chromofuscus PLD inhibitor, was measured by means of the infrared assay, demonstrating that this assay can be used to screen PLD activity and/or the specificity of its inhibitors.     

The diversity of algal phospholipase D homologs revealed by biocomputational analysis

Phospholipase D (PLD) participates in the formation of phosphatidic acid, a precursor in glycerolipid biosynthesis and a second messenger. PLDs are part of a superfamily of proteins that hydrolyze phosphodiesters and share a catalytic motif, HxKxxxxD, and hence a mechanism of action. Although HKD‐PLDs have been thoroughly characterized in plants, animals and bacteria, very little is known about these enzymes in algae. To fill this gap in knowledge, we performed a biocomputational analysis by means of HMMER iterative profiling, using most eukaryotic algae genomes available. Phylogenetic analysis revealed that algae exhibit very few eukaryotic‐type PLDs but possess, instead, many bacteria‐like PLDs. Among algae eukaryotic‐type PLDs, we identified C2‐PLDs and PXPH‐like PLDs. In addition, the dinoflagellate Alexandrium tamarense features several proteins phylogenetically related to oomycete PLDs. Our phylogenetic analysis also showed that algae bacteria‐like PLDs (proteins with putative PLD activity) fall into five clades, three of which are novel lineages in eukaryotes, composed almost entirely of algae. Specifically, Clade II is almost exclusive to diatoms, whereas Clade I and IV are mainly represented by proteins from prasinophytes. The other two clades are composed of mitochondrial PLDs (Clade V or Mito‐PLDs), previously found in mammals, and a subfamily of potentially secreted proteins (Clade III or SP‐PLDs), which includes a homolog formerly characterized in rice. In addition, our phylogenetic analysis shows that algae have non‐PLD members within the bacteria‐like HKD superfamily with putative cardiolipin synthase and phosphatidylserine/phosphatidylglycerophosphate synthase activities. Altogether, our results show that eukaryotic algae possess a moderate number of PLDs that belong to very diverse phylogenetic groups.     

Binding of proteolytically processed phospholipase D from Streptomyces chromofuscus to phosphatidylcholine membranes facilitates vesicle aggregation and fusion.

Ca(2+)-dependent phospholipase D is secreted from Streptomyces chromofuscus as an intact enzyme of 57 kDa (PLD(57)). Under certain growth conditions, PLD is proteolytically cleaved and activated to form PLD(42/20) (named for the apparent size of the peptides). The PLD(42) catalytic core and 20 kDa C-terminal domain remain tightly associated through noncovalent interactions. In the presence of Ba(2+) (to enhance protein binding to zwitterionic vesicles without hydrolysis of substrate), PLD(42/20), but not PLD(57), induces POPC vesicle leakiness as measured by entrapped CF leakage. PLD(42/20) also induces vesicle fusion (as measured by light scattering, fluorescence quenching, and cryo-TEM) under these conditions (1 mM POPC, 5 mM Ba(2+)); neither PLD(42) nor PLD(20) alone can act as a fusogen. For intact PLD(57) to cause CF leakiness, the soluble activator diC(4)PA must be present. However, even with diC(4)PA, PLD(57) does not induce significant vesicle fusion. In the absence of metal ions, all PLD forms bind to PC vesicles doped with 10 mol % PA. Again, only PLD(42/20) is fusogenic and causes aggregation and fusion on a rapid time scale. Taken together, these data suggest that activated PLD(42/20) inserts more readily into the lipid bilayer than other PLD forms and creates structures that allow bilayers to fuse. Cleavage of the PLD(57) by a secreted protease to generate PLD(42/20) occurs in the late stages of S. chromofuscus cell cultures. Production of this more active and fusogenic enzyme may play a role in nutrient scavenging in stationary phase cultures.     

The transphosphatidylation potential of a membrane-bound phospholipase D from poppy seedlings

Plant phospholipases D (PLDs) occur in a large variety of isoenzymes, which differ in Ca(2+) ion requirement, phosphatidylinositol-4,5-bisphosphate (PIP(2)) activation and substrate selectivity. In the present study a membrane-bound PLD has been identified in the microsomal fractions of poppy seedlings (Papaver somniferum). The maximum PLD activity is found after 2 days of germination in endosperms and after 3 days in developing seedlings. In contrast to the four poppy PLD isoenzymes described hitherto, the membrane-bound form is active at lower Ca(2+) ion concentrations (in the micromolar instead of millimolar range) and needs PIP(2) for hydrolytic activity. Remarkable differences are also observed in head group exchange reactions. The reaction rates of the transphosphatidylation of phosphatidylcholine by various acceptor alcohols follow the sequence glycerol>serine>myo-inositol>ethanolamine, whereas ethanolamine is preferred by most other PLDs. Despite the biocatalytic differences, the membrane-bound PLD interacts with polyclonal antibodies raised against α-type PLD, which reveals some structural similarities between these two enzymes.     

The Arabidopsis phospholipase D family. Characterization of a calcium-independent and phosphatidylcholine-selective PLD zeta 1 with distinct regulatory domains

Four types of phospholipase D (PLD), PLD alpha, beta, gamma, and delta, have been characterized in Arabidopsis, and they display different requirements for Ca(2+), phosphatidylinositol 4,5-bisphosphate (PIP(2)), substrate vesicle composition, and/or free fatty acids. However, all previously cloned plant PLDs contain a Ca(2+)-dependent phospholipid-binding C2 domain and require Ca(2+) for activity. This study documents a new type of PLD, PLD zeta 1, which is distinctively different from previously characterized PLDs. It contains at the N terminus a Phox homology domain and a pleckstrin homology domain, but not the C2 domain. A full-length cDNA for Arabidopsis PLD zeta 1 has been identified and used to express catalytically active PLD in Escherichia coli. PLD zeta 1 does not require Ca(2+) or any other divalent cation for activity. In addition, it selectively hydrolyzes phosphatidylcholine, whereas the other Arabidopsis PLDs use several phospholipids as substrates. PLD zeta 1 requires PIP(2) for activity, but unlike the PIP(2)-requiring PLD beta or gamma, phosphatidylethanolamine is not needed in substrate vesicles. These differences are described, together with a genomic analysis of 12 putative Arabidopsis PLD genes that are grouped into alpha, beta, delta, gamma, and zeta based on their gene architectures, sequence similarities, domain structures, and biochemical properties.     

Extending the C2 domain family: C2s in PKCs delta, epsilon, eta, theta, phospholipases, GAPs, and perforin.

Various membrane lipid metabolites, generated by phospholipases C and D (PLCs, PLDs), are known to regulate the activities of protein kinases C (PKCs) and GTP-ase activating proteins (GAPs) in a range of cellular processes. Conventional Ca(2+)-dependent PKCs (alpha, beta I, beta II, and gamma), PLCs and various GAPs are all known to contain copies of a phospholipid-binding domain, termed C2 or CalB. Here we recognize that C2 domains are also present in "new" Ca(2+)-independent PKCs (delta, epsilon, eta, and theta), other kinases, a eukaryotic PLD, the breakpoint cluster region (BCR) gene product, and two further GAPS. Twenty-two previously unrecognized C2 domain sequences are presented, which include a single copy in the mammalian poreforming proteins, perforin.     

The role of interfacial binding in the activation of Streptomyces chromofuscus phospholipase D by phosphatidic acid

The Streptomyces chromofuscus phospholipase D (PLD) cleavage of phosphatidylcholine in bilayers can be enhanced by the addition of the product phosphatidic acid (PA). Other anionic lipids such as phosphatidylinositol, oleic acid, or phosphatidylmethanol do not activate this PLD. This allosteric activation by PA could involve a conformational change in the enzyme that alters PLD binding to phospholipid surfaces. To test this, the binding of intact PLD and proteolytically cleaved isoforms to styrene divinylbenzene beads coated with a phospholipid monolayer and to unilamellar vesicles was examined. The results indicate that intact PLD has a very high affinity for PA bilayers at pH >/= 7 in the presence of EGTA that is weakened as Ca(2+) or Ba(2+) are added to the system. Proteolytically clipped PLD also binds tightly to PA in the absence of metal ions. However, the isolated catalytic fragment has a considerably weaker affinity for PA surfaces. In contrast to PA surfaces, all PLD forms exhibited very low affinity for PC interfaces with an increased binding when Ba(2+) was added. All PLD forms also bound tightly to other anionic phospholipid surfaces (e.g. phosphatidylserine, phosphatidylinositol, and phosphatidylmethanol). However, this binding was not modulated in the same way by divalent cations. Chemical cross-linking studies suggested that a major effect of PLD binding to PA.Ca(2+) surfaces is aggregation of the enzyme. These results indicate that PLD partitioning to phospholipid surfaces and kinetic activation are two separate events and suggest that the Ca(2+) modulation of PA.PLD binding involves protein aggregation that may be the critical interaction for activation.     

S100P, a novel Ca(2+)-binding protein from human placenta. cDNA cloning, recombinant protein expression and Ca2+ binding properties.

A novel member of the S100 protein family, present in human placenta, has been characterized by protein sequencing, cDNA cloning, and analysis of Ca(2+)-binding properties. Since the placenta protein of 95 amino acid residues shares about 50% sequence identity with the brain S100 proteins alpha and beta, we proposed the name S100P. The cDNA was expressed in Escherichia coli and recombinant S100P was purified in high yield. S100P is a homodimer and has two functional EF hands/polypeptide chain. The low-affinity site (Kd = 800 microM), which, in analogy to S100 beta, seems to involve the N-terminal EF hand, can be followed by the Ca(2+)-dependent decrease in tyrosine fluorescence. The high-affinity site, provided by the C-terminal EF hand, influences the reactivity of the sole cysteine which is located in the C-terminal extension (Cys85). Binding to the high-affinity site (Kd = 1.6 microM) can be monitored by fluorescence spectroscopy of S100P labelled at Cys85 with 6-proprionyl-2-dimethylaminonaphthalene (Prodan). The Prodan fluorescence shows a Ca(2+)-dependent red shift of the maximum emission wavelength from 485 nm to 502 nm, which is accompanied by an approximately twofold loss in integrated fluorescence intensity. This indicates that Cys85 becomes more exposed to the solvent in Ca(2+)-bound S100P, making this region of the molecule, the so-called C-terminal extension, an ideal candidate for a putative Ca(2+)-dependent interaction with a cellular target. In p11, a different member of the S100 family, the C-terminal extension which contains a corresponding cysteine (Cys82 in p11), is involved in a Ca(2+)-independent complex formation with the protein ligand annexin II. The combined results support the hypothesis that S100 proteins interact in general with their targets after a Ca(2+)-dependent conformational change which involves hydrophobic residues of the C-terminal extension.