Concanavalin A will not assume the sugar binding conformation in the complete absence of metal ions

Concanavalin A (Con A) is known to exist in two conformations that differ in their capacity to bind metals and sugars. The conformation that binds metals tightly and has a high affinity for sugars is termed the “locked” form and the conformation with low affinity for sugars and binds metals weakly is termed the “unlocked” form. It has recently been reported [Brown, $\text{et al.}$ Biochemistry $\text{21}$, 465(1982)] that apo-Con A will form the locked conformation in the absence of metals to the extent of 12.5% when equilibrated at 25°C for one week. In this report we show that Con A will not assume the locked conformation in the complete absence of metals and that only trace amounts of Ca²⁺ can catalytically convert a significant amount of the protein into the locked conformation.     

Metal ion induced conformational changes in concanavalin A: evidence for saccharide binding to one metal free structure.

The addition of Mn²⁺, Zn²⁺, Co²⁺, Ca²⁺ or Pb²⁺ to apo-concanavalin A results in a slow conformational conversion of the protein to the active saccharide binding form. The rates of conversion are dependent upon the sample pH and identity of the ions which occupy the native transition metal and calcium ion sites yet the affinity of each metalloform for the fluorescent sugar, 4-methylumbelliferyl-α-D-mannopyranoside, is independent of these same parameters (above pH 5.6). EDTA quickly removes all metal ions from the active Mn²⁺ or Co²⁺-concanavalin A samples leaving a metastable metal free structure which retains its high saccharide affinity for several hours at room temperature. This form of apo-concanavalin A and the metallized derivatives have equally high saccharide binding affinities in 1M NaCL but the former dramatically loses its sugar affinity as the ionic strength is lowered.     

Kinetic studies of the demetallization and inactivation of concanavalin A

The demetallization of various metallo derivatives of Concanavalin A (i.e., MnMnPL, CoMnPL, CaCaPL, CoCaPL and MnCaPL, where PL represents protein in a locked conformation) has been examined by three separate procedures. These include the treatment of the protein with the metal ion chelators, EDTA and terpyridine, and subjecting the protein to low pH (i.e., pH 1.2). In all three procedure and for all five species examined, the immediate product of protein demetallization was the PL conformation previously described by Brown, R.D., III, Brewer, C.F. and Koenig, S.H. (Biochemistry (1977) 16, 3883-3896). The rates of dissociation of the metals from the different protein species, as measured spectrophotometrically using terpyridine, were found to be identical to the rates (k1) of loss of protein sugar binding affinity in the presence of EDTA as measured by assays with the fluorescent sugar, 4-methylumbelliferyl alpha-D-mannoside. The kinetic and thermodynamic data associated with the inactivation of the protein species have allowed the different metallo derivatives to be classed into two general categories. Class I forms include MnMnPL, CoMnPL and CaCaPL and possess an average k1 (25 degrees C) value of 3.88 X 10(-2) s-1 and an average Ea of 14.2 kcal X mol-1. Class II forms CoCaPL and MnCaPL have average values for k1 (25 degrees C) and Ea of 3.67 X 10(-5) s-1 and 21.6 kcal X mol-1, respectively.     

Spectroscopic studies on the protective effect of a specific sugar on concanavalin A at acidic, neutral and alkaline pH

A Systematic investigation of the effect of pH on concanavalin A in the presence of specific and non-specific sugars is made using CD (circular dichroism) and fluorescence. The specific and non-specific sugars for concanavalin A were methyl alpha-D-glucopyranoside and methyl alpha-D-galactopyranoside respectively. Far-UV CD showed changes in the MRE value at 217 nm in the presence of the above-mentioned sugars. At pH 7, the CD and fluorescence spectra obtained in the presence of methyl alpha-D-glucopyranoside were slightly different from those for the native state and a significant difference was obtained in the presence of methyl alpha-D-galactopyranoside. Near-UV CD spectra showed the retention of a native-like tertiary structure in the presence of the specific sugar upon pH denaturation. Tryptophan fluorescence studies indicated a change in the tryptophan enviornment. The results obtained from our CD data are consistent with those obtained from fluorescence studies. Upon pH exposure of concanavalin A in the presence of methyl alpha-D-glucopyranoside and methyl alpha-D-galactopyranoside, the former acted as a protector preventing conformational alteration at different pH while the presence of latter induced a different stable conformational state and this state persists over the pH range from 2 to 10.     

Redox-dependent conformational selection in a Cys4Fe2S2 ferredoxin

Putidaredoxin (Pdx), a Cys4Fe2S2 ferredoxin from Pseudomonas putida, exhibits redox-dependent binding to its physiological redox partner, cytochrome P450(cam) (CYP101), with the reduced form of Pdx (Pdx(r)) binding with greater affinity to oxidized camphor-bound CYP101 than the oxidized form, Pdx(o). It has been previously shown that Pdx(o) is more dynamic than Pdx(r) on all accessible time scales, and it has been proposed that Pdx(r) samples only a fraction of the conformational substates populated by Pdx(o) on a time average. It is postulated that the ensemble subset populated by Pdx(r) is the same subset that binds CYP101, providing a mechanism for coupling the Pdx oxidation state to binding affinity for CYP101. Evidence from a variety of sources, including redox-dependent shifts of 15N and 13C resonances, indicates that the metal cluster binding loop of Pdx is the primary determinant of redox-dependent conformational selection. Patterns of paramagnetic effects suggest that the metal cluster binding loop contracts around the metal cluster upon reduction, possibly due to the strengthening of hydrogen bonds between the sulfur atoms of the metal cluster and the surrounding polypeptide NH and OH groups. Effects of this perturbation are then transmitted mechanically to other affected regions of the protein. A specific mutation has been introduced into the metal binding loop of Pdx, G40N, that slows conformational exchange sufficiently that the ensemble of conformational substates in Pdx(o) are directly observable as severe broadenings or splittings in affected NMR resonances. Many of the residues most affected by the mutation also show significant exchange contributions to 15N T(2) relaxation in wild-type Pdx(o). As predicted, G40N Pdx(r) shows a collapse of many of these multiplets and broadened lines to form much sharper resonances that are essentially identical to those observed in wild-type Pdx(r), indicating that Pdx(r) occupies fewer conformational substates than does Pdx(o). This is the first direct observation of such redox-dependent ensembles at slow exchange on the chemical shift time scale. These results confirm that conformational selection within the Fe2S2 cluster binding loop is the primary source of redox-dependent changes in protein dynamics in Pdx.     

Interplay between metal binding and cis/trans isomerization in legume lectins: structural and thermodynamic study of P. angolensis lectin

The interplay between metal binding, carbohydrate binding activity, stability and structure of the lectin from Pterocarpus angolensis was investigated. Removal of the metals leads to a more flexible form of the protein with significantly less conformational stability. Crystal structures of this metal-free form show significant structural rearrangements, although some structural features that allow the binding of sugars are retained. We propose that substitution of an asparagine residue at the start of the C-terminal beta-strand of the legume lectin monomer hinders the trans-isomerization of the cis-peptide bond upon demetallization and constitutes an intramolecular switch governing the isomer state of the non-proline bond and ultimately the lectin phenotype.     

Engineered disulfide reveals structural dynamics of locked SARS-CoV-2 spike

The spike (S) protein of SARS-CoV-2 has been observed in three distinct pre-fusion conformations: locked, closed and open. Of these, the function of the locked conformation remains poorly understood. Here we engineered a SARS-CoV-2 S protein construct “S-R/x3” to arrest SARS-CoV-2 spikes in the locked conformation by a disulfide bond. Using this construct we determined high-resolution structures confirming that the x3 disulfide bond has the ability to stabilize the otherwise transient locked conformations. Structural analyses reveal that wild-type SARS-CoV-2 spike can adopt two distinct locked-1 and locked-2 conformations. For the D614G spike, based on which all variants of concern were evolved, only the locked-2 conformation was observed. Analysis of the structures suggests that rigidified domain D in the locked conformations interacts with the hinge to domain C and thereby restrains RBD movement. Structural change in domain D correlates with spike conformational change. We propose that the locked-1 and locked-2 conformations of S are present in the acidic high-lipid cellular compartments during virus assembly and egress. In this model, release of the virion into the neutral pH extracellular space would favour transition to the closed or open conformations. The dynamics of this transition can be altered by mutations that modulate domain D structure, as is the case for the D614G mutation, leading to changes in viral fitness. The S-R/x3 construct provides a tool for the further structural and functional characterization of the locked conformations of S, as well as how sequence changes might alter S assembly and regulation of receptor binding domain dynamics.     

Effect of Cation Concentration on Molecular Dynamics Simulations of UDP-Glucose

Abstract

Glycosyl esters of nucleoside di or mono-phosphates, generally referred to as “sugar nucleotides”, serve as a sugar donors during the biosynthesis of oligo- and polysaccharides; they are therefore of a primary importance in carbohydrate metabolism in the living world. Molecular dynamics simulations were used to explore the conformational flexibility of one nucleotide sugar, UDP-glucose (UDP-Glc). The AMBER program package was used with some new parameters especially developed for nucleotide sugars. Several simulations on this molecule in aqueous solution, each of 2 ns duration, were carried out for increasing concentrations of monovalent K⁺ and divalent Mg²⁺ ions. For the monovalent ion, it is revealed that its presence and concentration is crucial for the conformational behavior, resulting in the stabilization of the extended conformation. The preferred location of K⁺ is in close proximity to the negatively charged phosphate oxygens, but the ion moves freely and can occupy other sites. Since the size of this cation is close that of the water molecules, the hydration scheme is not perturbed. Completely different results are obtained when the divalent Mg²⁺ cation is introduced in the simulation. A very strong interaction is established between the phosphate group and the cation; as a result the UDP-Glc molecule is locked in a rigid extended geometry. The analyses of the trajectories provide new insight on the role of the metal ion in the catalytic mechanism of glycosyltransferases.     

A Pro to Gly mutation in the hinge of the arabinose-binding protein enhances binding and alters specificity. Sugar-binding and crystallographic studies

The L-arabinose-binding protein (ABP) of Escherichia coli consists structurally of two distinct globular domains connected by a hinge of three separate peptide segments. Arabinose is bound and completely sequestered within the deep cleft between the two domains. With reduced affinity, ABP also binds D-galactose (approximately 2-fold reduction) and D-fucose (approximately 40-fold reduction). Experiments have been conducted to explore the role in sugar binding of the hinge connecting the two domains of ABP. To increase the flexibility of the hinge region, a glycine was substituted for a proline at position 254 by site-directed mutagenesis. Unexpectedly, this mutation resulted in the dramatic enhancement of galactose binding over that of arabinose. The affinity of the mutant ABP for galactose increased by over 20-fold, while that for arabinose and fucose remained relatively unchanged. We have measured association and dissociation rates of the Gly-254 ABP with L-arabinose, D-galactose, and D-fucose and have determined the crystallographic structure of the protein complexed with each of the three sugars. Both the ligand-binding kinetic measurements and structure analysis indicate that the altered specificity is due to an effective increase in the rigidity of the hinge in the closed conformation which is induced upon galactose binding. Stabilizing contacts are formed between the strands of the hinge in the Gly-254 ABP when galactose is bound which are not found in complexes with the other sugars or the liganded wild-type protein.     

Variable H+/substrate stoicheiometries in Rhodotorula gracilis are caused by a pH-dependent protonation of the carrier(s).

Two carrier-mediated systems transport sugars in the yeast Rhodotorula gracilis depending on the pH. One system, with higher affinity for sugars, catalyses a symport of protons with sugar, whereas the other system, having lower affinity, is independent of protons. This was shown in three different ways. (1) At low pH, where only the high-affinity system works, a H+/sugar stoicheiometry of 1 was found. An increase of the pH and of the sugar concentration, which allowed the low-affinity system to operate, brought about a drop of the stoicheiometry to values below 1. (2) During H+ symport the influx of positive charge was electrically compensated by an equivalent efflux of K+ from the cells. At high pH and high sugar concentrations this stoicheiometry of K+ and sugar decreased concomitant with the H+/sugar stoicheiometry. (3) At pH 7.5 both transport systems were operating, as shown by biphasic saturation kinetics. Under these conditions only the high-affinity transport was found to be electrogenic. These results agree with the theory of an electrogenic H+/sugar symport where changes in the affinity for substrate are brought about by reversible protonation and deprotonation of the carrier.     

Metal induced structural changes observed in hexameric insulin

The metal ions in insulin hexamer play a crucial role in the T to R conformational transitions. We have determined the crystal structures of 2Mn2+, 1Rb1+ and 4Ni2+ human arg-insulin and compared them with the 2Zn2+ structure. The first two structures exist in the T3R3f state like the native 2Zn2+ arg-insulin, while the 4Ni2+ adopts a T6 conformation. The metal coordination is found to be tetrahedral in all the structures except that of nickel where a dual octahedral and tetrahedral coordination is found at one site. Rubidium occupies only one of the high affinity metal binding sites. The metal induced structural changes observed, have been explained.     

Metal Ion Binding and Conformational Transitions in Concanavalin A: A Structure-Function Study

Abstract

The affinity of the lectin Concanavalin A (Con A) for saccharides, and its requirement for metal ions such as Mn²⁺ and Ca²⁺, have been known for about 50 years. However the relationship between metal ion binding and the saccharide binding activity of Con A has only recently been examined in detail. Brown et al. (Biochemistry 16, 3883 (1977)) showed that Con A exists as a mixture of two conformational states: a “locked” form and an “unlocked” form. The unlocked form of the protein weakly binds metal ions and saccharide, and is the predominate conformation of demetallized Con A (apo-Con A) at equilibrium. The locked form binds two metal ions per monomer with the resulting complex(es) possessing full saccharide binding activity. Brown and coworkers measured the kinetics of the transition of the unlocked form to the fully metallized locked conformation containing Mn²⁺and Ca²⁺. They also demonstrated that Mn²⁺ alone could form a locked ternary complex with Con A, and that rapid removal of the ions resulted in a metastable form of apo-Con A in the locked conformation which slowly (hours at 25°C) reverted back to (predominantly) the unlocked conformation. The ability to form either conformation in the absence or presence of metal ions has thus allowed us to explore the relationship between metal ion binding and conformational transitions in Con A as determinants of the saccharide binding activity of the lectin.

Based on the kinetics of the transition of unlocked apo-Con A to fully metallized locked Con A, and X-ray crystallographic data, it appears that the transition between the two conformations of Con A involves a cis-trans isomerization of an Ala-Asp peptide bond in the backbone of the protein, near one of the two metal ion binding sites. The relatively large activation energy for the transition (～ 22 kcal M^?1) results in relatively slow interconversions between the conformations (from minutes to days), whereas the equilibria with metal ions and saccharide are rapid. Thus, many metastable complexes can be formed and a variety of transition pathways between the two conformations studied.

We have identified and characterized binary, ternary, and quaternary complexes of both conformations of Con A containing Mn²⁺ and saccharide, and have determined both metalion and saccharide dissociation constants for all of them, as well as equilibrium and kinetic values for the conformational transitions between them. The main finding is that saccharide binds very weakly (K_d～2 M) to unlocked apo-Con A and very tightly to the locked ternary Mn²⁺-Con A complex (K_d～ 10^?4 M). Saccharide binding increases along the various pathways connecting these two species in a nonadditive fashion. Thus, both conformation and metal ion binding determine the saccharide affinity of each complex, although the specificity of saccharide binding of the various species is maintained throughout.     

The crystal structure of metal-free human EF-hand protein S100A3 at 1.7-A resolution

S100A3 is a unique member of the EF-hand superfamily of Ca(2+)-binding proteins. It binds Ca(2+) with poor affinity (K(d) = 4-35 mm) but Zn(2+) with exceptionally high affinity (K(d) = 4 nm). This high affinity for Zn(2+) is attributed to the unusual high Cys content of S100A3. The protein is highly expressed in fast proliferating hair root cells and astrocytoma pointing toward a function in cell cycle control. We determined the crystal structure of the protein at 1.7 A. The high resolution structure revealed a large distortion of the C-terminal canonical EF-hand, which most likely abolishes Ca(2+) binding. The crystal structure of S100A3 allows the prediction of one putative Zn(2+) binding site in the C terminus of each subunit of S100A3 involving Cys and His residues in the coordination of the metal ion. Zn(2+) binding induces a large conformational change in S100A3 perturbing the hydrophobic interface between two S100A3 subunits, as shown by size exclusion chromatography and CD spectroscopy.     

Ions binding to S100 proteins. I. Calcium- and zinc-binding properties of bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) protein: Zn2+ regulates Ca2+ binding on S100b protein

Flow dialysis measurements of calcium binding to bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) proteins in 20 mM Tris-HCl buffer at pH 7.5 and 8.3 revealed that S100 proteins bind specifically 4 Ca2+ eq/mol of protein dimer. The specific calcium-binding sites had, therefore, been assigned to typical amino acid sequences on the alpha and beta subunit. The protein affinity for calcium is much lower in the presence of magnesium and potassium. Potassium strongly antagonizes calcium binding on two calcium-binding sites responsible for most of the Ca2+-induced conformational changes on S100 proteins (probably site II alpha and site II beta). Zinc-binding studies in the absence of divalent cations revealed eight zinc-binding sites/mol of S100b protein dimer that we assumed to correspond to 4 zinc-binding sites/beta subunit. Zinc binding to S100b studied with UV spectroscopy methods showed that the occupation of the four higher affinity sites and the four lower affinity sites on the protein dimer were responsible for different conformational changes in S100b structure. Zinc binding on the higher affinity sites regulates calcium binding to S100b by increasing the protein affinity for calcium and decreasing the antagonistic effect of potassium on calcium binding. Zinc-binding studies on S100a and S100 alpha alpha protein showed that the Trp-containing S100 proteins bind zinc more weakly than S100b protein. Calcium-binding studies on zinc-bound S100a proved that calcium- and zinc-binding sites were distinct although there was no increase in zinc-bound S100a affinity for calcium, as in S100b protein. Finally we provide evidence that discrepancies between previously published results on the optical properties of S100b protein probably result from oxidation of the sulfhydryl groups in the protein.     

Conformational and thermodynamic properties of peptide binding to the human S100P protein

S100P is a member of the S100 subfamily of calcium-binding proteins that are believed to be associated with various diseases, and in particular deregulation of S100P expression has been documented for prostate and breast cancer. Previously, we characterized the effects of metal binding on the conformational properties of S100P and proposed that S100P could function as a Ca2+ conformational switch. In this study we used fluorescence and CD spectroscopies and isothermal titration calorimetry to characterize the target-recognition properties of S100P using a model peptide, melittin. Based on these experimental data we show that S100P and melittin can interact in a Ca2+-dependent and -independent manner. Ca2+-independent binding occurs with low affinity (Kd approximately 0.2 mM), has a stoichiometry of four melittin molecules per S100P dimer and is presumably driven by favorable electrostatic interactions between the acidic protein and the basic peptide. In contrast, Ca2+-dependent binding of melittin to S100P occurs with high affinity (Kd approximately 5 microM) has a stoichiometry of two molecules of melittin per S100P dimer, appears to have positive cooperativity, and is driven by hydrophobic interactions. Furthermore, Ca2+-dependent S100P-melittin complex formation is accompanied by significant conformational changes: Melittin, otherwise unstructured in solution, adopts a helical conformation upon interaction with Ca2+-S100P. These results support a model for the Ca2+-dependent conformational switch in S100P for functional target recognition.     

Magnesium dependence of the amplified conformational switch in the trans-acting hepatitis delta virus ribozyme

The ability of divalent metal ions to participate in both structure formation and catalytic chemistry of RNA enzymes (ribozymes) has made it difficult to separate their cause and effect in ribozyme function. For example, the recently solved crystal structures of precursor and product forms of the cis-cleaving genomic hepatitis delta virus (HDV) ribozyme show a divalent metal ion bound in the active site that is released upon catalysis due to an RNA conformational change. This conformational switch is associated with a repositioning of the catalytically involved base C75 in the active-site cleft, thus controlling catalysis. These findings confirm previous data from fluorescence resonance energy transfer (FRET) on a trans-acting form of the HDV ribozyme that found a global conformational change to accompany catalysis. Here, we further test the conformational switch model by measuring the Mg(2+) dependence of the global conformational change of the trans-acting HDV ribozyme, using circular dichroism and time-resolved FRET as complementary probes of secondary and tertiary structure formation, respectively. We observe significant differences in both structure and Mg(2+) affinity of the precursor and product forms, in the presence and absence of 300 mM Na(+) background. The precursor shortens while the product extends with increasing Mg(2+) concentration, essentially amplifying the structural differences observed in the crystal structures. In addition, the precursor has an approximately 2-fold and approximately 13-fold lower Mg(2+) affinity than the product in secondary and tertiary structure formation, respectively. We also have compared the C75 wild-type with the catalytically inactive C75U mutant and find significant differences in global structure and Mg(2+) affinity for both their precursor and product forms. Significantly, the Mg(2+) affinity of the C75 wild-type is 1.7-2.1-fold lower than that of the C75U mutant, in accord with the notion that C75 is essential for a catalytic conformational change that leads to a decrease in the local divalent metal ion affinity and release of a catalytic metal. Thus, a consistent picture emerges in which divalent metal ions and RNA functional groups are intimately intertwined in affecting structural dynamics and catalysis in the HDV ribozyme.     

Conformational changes in Sindbis virus induced by decreased pH are revealed by small-angle neutron scattering

Alphaviruses, such as Sindbis virus, undergo dramatic changes in three-dimensional structure upon exposure to low pH, and such exposure can establish conditions allowing fusion of the virus membrane with a cell plasma membrane upon return to neutral pH. While exposure to low pH is not required for entry of Sindbis virus into vertebrate or invertebrate cells, the conformational changes occurring at low pH may mimic those occurring upon virus-receptor interaction. Here, we employed small-angle neutron scattering with contrast variation to probe how the structure of a mammalian-grown Sindbis virus responds to moderately acidic pH. Several changes took place throughout the virion structure when the pH decreased from 7.2 to 6.4. Specifically, the RNA in the virion core underwent a conformational change. Additionally, the protein was redistributed. A significant amount of protein moved from the layer containing the lipid bilayer to the exterior of the virion. The results improve our understanding of the pH-driven alteration of Sindbis virus structure.     

Understanding the binding interaction between phenyl boronic acid P1 and sugars: determination of association and dissociation constants using S–V plots,steady‐state spectroscopic methods and molecular docking

Continuous monitoring of glucose and sugar sensing plays a vital role in diabetes control. The drawbacks of the present enzyme‐based sugar sensors have encouraged the investigation into alternate approaches to design new sensors. The popularity of fluorescence sensors is due to their ability to bind reversibly to compounds containing diol. In this study we investigated the binding ability of phenyl boronic acid P1 for monosaccharides and disaccharides (sugars) in aqueous medium at physiological pH 7.4 using steady‐state fluorescence and absorbance. P1 fluorescence was quenched due to formation of esters with sugars. Absorbance and fluorescence measurements led to results that indicated that the sugars studied could be ordered in terms of their affinity to P1, as stated: sucrose > lactose > galactose > xylose > ribose > arabinose. In each case, the slope of modified Stern–Volmer plots was nearly 1, indicating the presence of only a single binding site in boronic acids for sugars. Docking studies were carried out using Schrodinger Maestro v.11.2 software. The binding affinity of phenyl boronic acid P1 with periplasmic protein (PDB ID 2IPM and 2IPL) was estimated using GlideScore.     

Binding of transition metals to S100 proteins

The S100 proteins are a unique class of EF-hand Ca²⁺ binding proteins distributed in a cell-specific, tissue-specific, and cell cycle-specific manner in humans and other vertebrates. These proteins are distinguished by their distinctive homodimeric structure, both intracellular and extracellular functions, and the ability to bind transition metals at the dimer interface. Here we summarize current knowledge of S100 protein binding of Zn²⁺, Cu²⁺ and Mn²⁺ ions, focusing on binding affinities, conformational changes that arise from metal binding, and the roles of transition metal binding in S100 protein function.