Coordinative binding of divalent cations with ligands related to bacterial spores. Equilibrium studies

It has been repeatedly postulated that the high heat resistance of bacterial spores is due to stabilization of biopolymers in the spore interior by a solid deposit of protective cement consisting of coordination complexes of ligands with divalent metal ions. This report presents data on metal-binding characteristics of some of the ligands related to spores as determined by means of potentiometric equilibrium measurements under conditions of temperature and ionic strength (t = 25.0°C; μ = 1.0 KNO₃) identical with those reported earlier by the authors in order to facilitate correlation by using comparable data. The spore ligands investigated in this study included 2,6-pyridinedicarboxylic acid (DPA), α,ε-diaminopimelic acid, D-glutamic acid, and D-alanine in a ratio of 1:1 with metal ions which are known to play a role in heat resistance of spores. Stability constants of the chelates of these spore ligands with metal ions such as Ca(II), Mg(II), Cu(II), Ni(II), Zn(II), Co(II), and Mn(II) have been determined. In general the metal chelates of DPA exhibited the greatest stability. On the basis of a consideration of the stability data together with the known configurations of the ligand and the coordination requirements of the metal ions, possible structures indicating the coordinate binding of the spore ligands with the metal ions are presented. All the metal chelates except those of Ca(II) were found to undergo hydrolysis and separation of solid phase in the pH range 7-8.5. The relatively greater hydrolytic stability of Ca(II) chelates and the high affinity of DPA for metal ions appear to be of biological significance insofar as these two spore components are more widely associated with the heat resistance of bacterial spores.     

Structure and function of the methionine aminopeptidases

The removal of the N-terminal methionine from proteins and peptides is dependent upon a novel class of proteases typified by the dinuclear metalloenzyme methionine aminopeptidase from Escherichia coli (eMetAP). Substantial progress has recently been made in determining the structures of several members of this family. The identification of human MetAP as the target of putative anti-cancer drugs reiterates the importance of this family of enzymes. Determination of the modes of binding to E. coli MetAP of a substrate-like bestatin-based inhibitor, as well as phosphorus-containing transition-state analogs and reaction products has led to a rationalization of the substrate specificity and suggested the presumed catalytic mechanism. The conservation of key active site residues and ligand interactions between the MetAPs and other enzyme of the same fold suggest that avoidance of cross-reactivity may be an important consideration in the design of inhibitors directed toward a single member of the family.     

Functional studies of the bacterial avirulence protein AvrPto by mutational analysis

Pseudomonas syringae pathovars expressing avrPto are avirulent on plants expressing the resistance gene Pto. Over 85 mutants of avrPto were generated with multiple strategies, and several assays were used to characterize AvrPto function. Only a core of 95 amino acids of the 164 residues was sufficient for binding Pto in the yeast two-hybrid system. Only nine of 65 mutant proteins of AvrPto with amino acid substitutions, created in planta and in vitro, did not interact with Pto in the Gal4 yeast two-hybrid system, suggesting that AvrPto can tolerate many nonconservative substitutions and still interact with Pto. These nine and 12 additional substitution mutants of AvrPto were characterized further. The ability to elicit a hypersensitive response and the effect on pathogenesis in planta for these 21 mutants of AvrPto were strongly correlated with recognition by Pto in the yeast two-hybrid system. Analyses of two proteins with substitutions H54P or D52G/L65P indicated that these residues may be required for delivery into the host cell and protein stability in the bacterial cytoplasm, respectively. The mutants that no longer interacted with Pto and had modified activities in planta were predicted to have changes in their secondary structure.     

S1 pocket fingerprints of human and bacterial methionine aminopeptidases determined using fluorogenic libraries of substrates and phosphorus based inhibitors

Methionyl aminopeptidases (MetAPs) are metallo-dependent proteases responsible for removing of N-terminal methionine residue of peptides and proteins during protein maturation and activation. In this report we use a comprehensive strategy to screen the substrate specificity of three methionyl aminopeptidases: Homo sapiens MetAP-1, Homo sapiens MetAP-2 and Escherichia coli MetAP-1. By utilizing a 65-membered fluorogenic substrate library consisting of natural and unnatural amino acids we established detailed substrate preferences of each enzyme in the S1 pocket. Our results show that this pocket is highly conserved for all investigated MetAPs, very stringent for methionine, and that several unnatural amino acids with methionine-like characteristics were also well hydrolyzed by MetAPs. The substrate-derived results were verified using several phosphonate and phosphinate-based inhibitors.     

Aggregation of nucleosomes by divalent cations.

Conditions of precipitation of nucleosome core particles (NCP) by divalent cations (Ca(2+) and Mg(2+)) have been explored over a large range of nucleosome and cation concentrations. Precipitation of NCP occurs for a threshold of divalent cation concentration, and redissolution is observed for further addition of salt. The phase diagram looks similar to those obtained with DNA and synthetic polyelectrolytes in the presence of multivalent cations, which supports the idea that NCP/NCP interactions are driven by cation condensation. In the phase separation domain the effective charge of the aggregates was determined by measurements of their electrophoretic mobility. Aggregates formed in the presence of divalent cations (Mg(2+)) remain negatively charged over the whole concentration range. They turn positively charged when aggregation is induced by trivalent (spermidine) or tetravalent (spermine) cations. The higher the valency of the counterions, the more significant is the reversal of the effective charge of the aggregates. The sign of the effective charge has no influence on the aspect of the phase diagram. We discuss the possible reasons for this charge reversal in the light of actual theoretical approaches.     

Insights into the activation of brain serine racemase by the multi-PDZ domain glutamate receptor interacting protein, divalent cations and ATP

Brain serine racemase contains pyridoxal phosphate as a prosthetic group and is known to become activated by divalent cations such as Ca(2+) and Mg(2+), as well as by ATP and ADP. In vivo, brain serine racemase is also activated by a multi-PSD-95/discs large/ZO-1 (PDZ) domain glutamate receptor interacting protein (GRIP) that is usually coupled to the GluR2/3 subunits of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid Ca(2+) channel. In the present study, we analysed the mechanisms by which serine racemase becomes activated by GRIP, divalent cations and ATP. We show that binding of PDZ6 of GRIP to serine racemase does not result in increased d-serine production. However, full-length GRIP does augment significantly enzymatic activity. We expressed various GRIP shorter constructs to map down the regions within GRIP that are necessary for serine racemase activation. We observed that, whereas recombinant proteins containing PDZ4-PDZ5-PDZ6 are unable to activate serine racemase, other constructs containing PDZ4-PDZ5-PDZ6-GAP2-PDZ7 significantly augment its activity. Hence, activation of serine racemase by GRIP is not a direct consequence of the translocation towards the calcium channel but rather a likely conformational change induced by GRIP on serine racemase. On the other hand, the observed activation of serine racemase by divalent cations has been assumed to be a side-effect associated with ATP binding, which is known to form a complex with Mg(2+) ions. Because no mammalian serine racemase has yet been crystallized, we used molecular modelling based on yeast and bacterial homologs to demonstrate that the binding sites for Ca(2+), ATP and the PDZ6 domain of GRIP are spatially separated and modulate the enzyme through distinct mechanisms.     

Regulation of the fibronectin receptor affinity by divalent cations

The cell surface receptor for fibronectin is a heterodimeric membrane protein that recognizes an Arg-Gly-Asp sequence in fibronectin and that requires cations such as Mg2+ or Ca2+ for binding to fibronectin. The divalent cation requirements of this receptor were analyzed by measuring attachment of receptor liposomes to ligand-coated surfaces in the presence of different cations. The most striking effect observed was produced by Mn2+, which increased the binding of the receptor liposomes to fibronectin 2-3-fold over their binding in buffers containing Ca2+ and Mg2+. The binding activities of two related adhesion receptors, the vitronectin receptor and platelet GP IIb-IIIa, were supported but not enhanced by Mn2+. Two observations suggest that Mn2+ can compete with Ca2+ for the same cation-binding sites of the receptor. First, Mn2+ could still enhance fibronectin receptor binding activity even in the presence of 10-fold higher concentrations of Ca2+ or Mg2+. Second, Mn2+ inhibited the binding of radioactive Ca2+ to the alpha subunit of the receptor. The increased fibronectin receptor activity in the presence of Mn2+ appeared to be due to an increase in the affinity of the receptor for the Arg-Gly-Asp sequence because a 110-kDa cell attachment fragment and a synthetic hexapeptide containing the Arg-Gly-Asp sequence inhibited liposome binding more effectively in the presence of Mn2+ than in the presence of Ca2+/Mg2+. The affinity for the peptide was affected more than the affinity for the fragment, indicating that Mn2+ also induces a change in receptor specificity. Increased receptor binding in the presence of Mn2+ was also apparent in affinity chromatography of the fibronectin receptor on the 110-kDa fibronectin fragment; Mn2+ improved the yield of the receptor 4-fold. Mn2+ similarly increased the number of receptor-fibronectin complexes in preparations analyzed by electron microscopy. These results show that exogenous influences can modulate the affinity and specificity with which the fibronectin receptor binds to its ligands.     

Structural basis for the functional differences between type I and type II human methionine aminopeptidases

Determination of the crystal structure of human MetAP1 makes it possible, for the first time, to compare the structures of a Type I and a Type II methionine aminopeptidase (MetAP) from the same organism. Comparison of the Type I enzyme with the previously reported complex of ovalicin with Type II MetAP shows that the active site of the former is reduced in size and would incur steric clashes with the bound inhibitor. This explains why ovalicin and related anti-angiogenesis inhibitors target Type II human MetAP but not Type I. The differences in both size and shape of the active sites between MetAP1 and MetAP2 also help to explain their different substrate specificity. In the presence of excess Co(2+), a third cobalt ion binds in the active site region, explaining why metal ions in excess can be inhibitory. Also, the N-terminal region of the protein contains three distinct Pro-x-x-Pro motifs, supporting the prior suggestion that this region of the protein may participate in binding to the ribosome.     

Modulation of linoleic acid-binding properties of human serum albumin by divalent metal cations

Human serum albumin (HSA) is an abundant multiligand carrier protein, linked to progression of Alzheimer’s disease (AD). Blood HSA serves as a depot of amyloid β (Aβ) peptide. Aβ peptide-buffering properties of HSA depend on interaction with its ligands. Some of the ligands, namely, linoleic acid (LA), zinc and copper ions are involved into AD progression. To clarify the interplay between LA and metal ion binding to HSA, the dependence of LA binding to HSA on Zn²⁺, Cu²⁺, Mg²⁺ and Ca²⁺ levels and structural consequences of these interactions have been explored. Seven LA molecules are bound per HSA molecule in the absence of the metal ions. Zn²⁺ binding to HSA causes a loss of one bound LA molecule, while the other metals studied exert an opposite effect (1–2 extra LA molecules are bound). In most cases, the observed effects are not related to the metal-induced changes in HSA quaternary structure. However, the Zn²⁺-induced decline in LA capacity of HSA could be due to accumulation of multimeric HSA forms. Opposite to Ca²⁺/Mg²⁺-binding, Zn²⁺ or Cu²⁺ association with HSA induces marked changes in its hydrophobic surface. Overall, the divalent metal ions modulate LA capacity and affinity of HSA to a different extent. LA- and Ca²⁺-binding to HSA synergistically support each other. Zn²⁺ and Cu²⁺ induce more pronounced changes in hydrophobic surface and quaternary structure of HSA and its LA capacity. A misbalanced metabolism of these ions in AD could modify interactions of HSA with LA, other fatty acids and hydrophobic substances, associated with AD.     

Regulation of smooth muscle phosphatase-II by divalent cations

Summary Smooth Muscle Phosphatases II (SMP-I1) which has been purified from turkey gizzards and previously classified as protein phosphatase 2C, is inactive in the absence of divalent cations. Study of the activation of SMP-II by Mg²⁺ and Mn²⁺ revealed differences in the modes of activation by these cations. The maximal activation elicited by Mg²⁺ is 1.5–2.5-fold higher than the maximal Mn²⁺ activation. However, the latter is achieved at a lower concentration than the maximal Mg²⁺-activation. Furthermore, at low cation concentrations ( 2 mM), the Mn²⁺-activated activity is higher than the Mg²⁺-activated activity. In the presence of both cations, the effect of Mn²⁺ predominates suggesting that the affinity of the enzyme for Mn²⁺ is greater than for Mg²⁺. In contrast to Mg²⁺ and Mn²⁺, Ca²⁺ does not activate SMP-II but it was observed to antagonize the effects of Mg²⁺ and Mn²⁺. Ca²⁺ acts as a competitive inhibitor of Mg²⁺. However, the inhibitory effect at high Ca²⁺ concentrations is not completely reversed by increasing the Mg²⁺ concentration. Mn²⁺ activation is also inhibited by Ca²⁺ but to a lesser extent. Ca²⁺ cannot completely inhibit Mn²⁺-activation suggesting that SMP-I1 has greater affinity for Mn²⁺ than for Ca²⁺. The finding that Ca²⁺ inhibits the activation of SMP-II raises the possibility that Ca²⁺ may be a regulator of SMP-II in vivo.Abbreviations SMP-II Smooth Muscle Phosphatase-II - MOPS 3-[N-Morpholine]propane Sulfonic Acid - PLC Phosphorylated Myosin Light Chains     

The regulation of integrin function by divalent cations

Integrins are a family of α/β heterodimeric adhesion metalloprotein receptors and their functions are highly dependent on and regulated by different divalent cations. Recently advanced studies have revolutionized our perception of integrin metal ion-binding sites and their specific functions. Ligand binding to integrins is bridged by a divalent cation bound at the MIDAS motif on top of either α I domain in I domain-containing integrins or β I domain in α I domain-less integrins. The MIDAS motif in β I domain is flanked by ADMIDAS and SyMBS, the other two crucial metal ion binding sites playing pivotal roles in the regulation of integrin affinity and bidirectional signaling across the plasma membrane. The β-propeller domain of α subunit contains three or four β-hairpin loop-like Ca²⁺-binding motifs that have essential roles in integrin biogenesis. The function of another Ca²⁺-binding motif located at the genu of α subunit remains elusive. Here, we provide an overview of the integrin metal ion-binding sites and discuss their roles in the regulation of integrin functions.     

The regulation of integrin function by divalent cations

Integrins are a family of α/β heterodimeric adhesion metalloprotein receptors and their functions are highly dependent on and regulated by different divalent cations. Recently advanced studies have revolutionized our perception of integrin metal ion-binding sites and their specific functions. Ligand binding to integrins is bridged by a divalent cation bound at the MIDAS motif on top of either α I domain in I domain-containing integrins or β I domain in α I domain-less integrins. The MIDAS motif in β I domain is flanked by ADMIDAS and SyMBS, the other two crucial metal ion binding sites playing pivotal roles in the regulation of integrin affinity and bidirectional signaling across the plasma membrane. The β-propeller domain of α subunit contains three or four β-hairpin loop-like Ca²⁺-binding motifs that have essential roles in integrin biogenesis. The function of another Ca²⁺-binding motif located at the genu of α subunit remains elusive. Here, we provide an overview of the integrin metal ion-binding sites and discuss their roles in the regulation of integrin functions.     

Chemical modification of low-density lipoprotein enhances the number of binding sites for divalent cations.

The EPR technique with paramagnetic Mn(II) ions has been used to probe the negatively charged sites on the surface of modified low-density lipoprotein (LDL). LDL modified in five different ways exhibited increased binding capacity for divalent cations. Enhanced binding is caused by the increase in the number of 'strong' binding sites. The 'strong' sites have been identified to be the aspartic acid and/or glutamic acid carboxyl residues and the 'weak' sites are zwitter-ionic phospholipids. In native LDL the negative groups make 'bonds' with the positive lysyl residues, thus stabilizing the structure. Any deprotonation or modification of the lysine amino groups makes the LDL structure more loose and the amino acid carboxyl groups accessible to divalent cations.     

The effect of divalent cations on the conformation and function of human plasminogen

The activation of native human plasminogen (Glu1-Pg) by tissue plasminogen activator, urinary plasminogen activator (u-PA), and streptokinase is inhibited by the divalent cations Ca2+, Mg2+, and Mn2+. This inhibition is accompanied by a conformational change in the molecule as evidenced by a decrease in Stokes' radius and intrinsic fluorescence. Kinetic analysis indicates that Mn2+ acts as an uncompetitive inhibitor of u-PA-catalyzed Glu1-Pg activation. In contrast to the inhibitory effects of divalent cations on Glu1-Pg, Ca2+ and Mg2+ stimulate the activation of proteolytically modified Lys77-Pg. These observations provide further evidence that Glu1-Pg and Lys77-Pg exhibit differential responses to ligands in the microenvironment.     

Further studies on the binding of divalent cations to the phosphoglycoprotein phosvitin.

Dialysis equilibrium measurements at 25 degrees indicate that, at pH 6.8 and at a concentration of 1.0 times 10(-10) 3 M MnC12 or CoC12, phosvitin binds 113 Mn2+ and 120 Co2+. The binding is cooperative at low cation concentrations. The number of Mg2+, Ca2+, Mn2+, and Co2+ bound is not affected by temperatures of up to 60 degrees; however, the cooperactivity is enhanced. Optical rotatory dispersion and circular dichroism studies indicate that a conformational change occurs on binding of Mn2+ and Co2+ which parallels the one produced by Ca2+ and reported elsewhere [Grizzuti, K., and Perlmann, G.E. (1973), Biochemistry 12, 4399]. The conformational changes induced by Mg2+ and Mn2+ follow different paths. Upon binding of Mn2+ and Co2+ the intrinsic viscosity, [eta], of phosvitin decreases from about 0.5 to 0.03 dl/g, while Mg2+ and Ca2+ decrease [eta] to 0.048 dl/g. The ultraviolet absorption spectrum of phosvitin is altered upon binding of Ca2+, Mn2+, and Co2+, but not upon binding of Mg2+; an increase of the temperature to 60% has no further effect on the spectra.