Why is dimerization important

Dimerization is believed to play a key role in the high thermal stability of TmDHFR, which is reflected in a melting temp. The dimer interface of TmDHFR is composed of a hydrophobic core with charged residues around the periphery. In particular, Lys of each subunit forms three-membered salt bridges with Glu and Glu of the other subunit. Our results indicate that these salt bridges are key for the high thermal stability of TmDHFR but are not a requirement for dimerization.

Although the rate of dihydrofolate redn. These changes are in agreement with the proposal that DHFR catalysis may be affected by changes to the conformational ensemble of the enzyme rather than only to the coupling of protein motions to the reaction coordinate.

Effect of Dimerization on Dihydrofolate Reductase Catalysis. Biochemistry , 52 , — , DOI: We investigated the effect of dimerization on DHFR catalysis by prepg. The steady-state and pre-steady-state rate consts. This redn. Portland Press Ltd. DHFR dihydrofolate reductase catalyzes the metabolically important redn. The turnover and the hydride-transfer rates in the kinetic scheme of TmDHFR were derived from measurements of the steady-state and pre-steady-state kinetics using absorbance and stopped-flow fluorescence spectroscopy.

The rate const. Hydride transfer was slow 0. Hydride transfer depended on ionization of a single group in the active site with a pKa of 6. Biochemistry , 36 , — , DOI: The reaction catalyzed by E.

The structures suggested that the M20 loop is predominantly closed over the reactants in the holoenzyme, Michaelis, and transition state complexes. However, during the remainder of the cycle, when nicotinamide is not bound, the loop occludes protrudes into the nicotinamide-ribose binding pocket.

The change may occur by way of an irregularly structured open loop conformation, which could transiently admit a water mol. From the Michaelis to the transition state analog complex, rotation between 2 halves of E.

The resulting enhancement of contacts with the pABG moiety may stabilize puckering at the C6 atom of the pteridine ring in the transition state. Subdomain rotation is not obsd. Loop movement, also unobsd. Academic Press. Two high-resoln. Whereas the overall fold of the hyperthermophilic enzyme was closely similar to monomeric mesophilic DHFR mols.

Here, the mol. The mol. Subunit cooperativity could be excluded from structural and biochem. Major contributions to the high intrinsic stability of the enzyme resulted from the formation of the dimer.

Within the monomer, only subtle stabilizing interactions were detectable, without clear evidence for any of the typical increments of thermal stabilization commonly reported for hyperthermophilic proteins.

The docking of the subunits was optimized with respect to high packing d. The enzyme did not show significant structural changes upon binding its coenzyme, NADPH, and the inhibitor, methotrexate. The active site loop, which is known to play an important role in catalysis in mesophilic DHFR, was rearranged, participating in the assocn. Biochemistry , 53 , — , DOI: Behiry, Enas M. Joel; Allemann, Rudolf K. Dihydrofolate reductase DHFR from Escherichia coli EcDHFR adopts 2 major conformations, closed and occluded, and the movement between these 2 conformations is important for progression through the catalytic cycle.

Destabilization of the occluded conformation did not affect hydride transfer but altered the affinity for NADP and changed the rate-detg. DHFRs from many organisms lack a H-bond donor in the appropriate position and hence most likely do not form an occluded conformation.

The link between conformational cycling between closed and occluded forms and progression through the catalytic cycle is specific to EcDHFR and not a general characteristic of prokaryotic DHFR catalysis.

Sequence detn. Ultracentrifugational anal. The enzyme exhibits long-term stability at physiol. The apoenzyme was crystd. Biochemistry , 46 , — , DOI: The possible relation between enzyme thermostability and catalytic power is of great current interest.

In particular, it has been suggested that thermophilic or hyperthermophilic Tm enzymes have lower catalytic power at a given temp. These suggestions presume that the reduced dynamics of the thermophilic enzymes is the reason for their reduced catalytic power.

The present paper takes the specific case of dihydrofolate reductase DHFR [of Escherichia coli Ms enzyme and Thermotoga maritima Tm enzyme ] and explores the validity of the above argument by simulation approaches.

It was found that Tm enzymes had restricted motions in the direction of the folding coordinate, but this was not relevant to the chem. Moreover, it was shown that the rate of the chem. In fact, as far as flexibility is concerned, it was concluded that the displacement along the reaction coordinate was larger in the Tm enzyme than in the Ms enzyme, and that the general trend in enzyme catalysis was that the best catalyst involved less motion during the reaction than the less optimal catalyst.

Thus, the relation between thermostability and catalysis appears to reflect the fact that in order to obtain small electrostatic reorganization energy it is necessary to invest some folding energy in the overall pre-organization process. Therefore, the optimized catalysts are less stable. This trend was clearly obsd. National Academy of Sciences. The structural origin of enzyme adaptation to low temp.

A remarkable universal fingerprint shared by all cold-active enzymes is a redn. Here, the authors explored the role of protein surface mobility in detg. The effects of modifying surface rigidity in cold- and warm-active trypsins were demonstrated here by calcn. The protein surface flexibility was systematically varied by applying positional restraints, causing the remarkable effect of turning the cold-active trypsin into a variant with mesophilic characteristics without changing the amino acid sequence.

Furthermore, the authors showed that just restraining a key surface loop caused the same effect as a point mutation in that loop between the cold- and warm-active trypsin. Computation of Enzyme Cold Adaptation. Simulations of hydride and deuteride transfer catalyzed by dihydrofolate reductase from the hyperthermophile Thermotoga maritima TmDHFR are presented. TmDHFR was modeled with its active homodimeric quaternary structure, where each monomer has three subdomains.

The potential energy function was a combined quantum mech. The calcns. The kinetic isotope effect was dominated by contributions of bound vibrations and decreased from 3. The calcd. The decreased catalytic efficiency of the monomer was therefore not the result of a decrease of the tunneling contribution but an increase in the quasi-classical activation free energy. The catalytic effect was assocd. The intrasubunit correlated motions were decreased in the monomer when compared to both native dimeric TmDHFR and monomeric E.

Science Reviews. Since the early days of enzymol. Here we present exptl. In this review, we first introduce basic concepts of enzyme catalysis from a phys.

Then, we present several recent developments in the application of exptl. These tools include kinetic isotope effects KIEs , their temp. Several theories and models that assist in understanding those phenomena are also described.

The possibility that these models invoke a direct role for the enzyme's dynamics environmental fluctuations and rearrangements is discussed. Finally, the need to compare the enzymic reaction to the uncatalyzed one while investigating contributions to catalysis is emphasized. Pure Appl. Seravalli, Javier; Huskey, W. Phillip; Schowen, K. Barbara; Schowen, Richard L. The lactate dehydrogenase of Bacillus stearothermophilus exists in dimeric and tetrameric forms, the latter favored by the regulatory effector, fructose-1,6-bisphosphate.

The kinetic behavior of the two forms is different, the tetramer showing activation relative to the dimer at low pyruvate concns. Deuterium isotope effects at the transferring hydride site of NADH primary isotope effect and the Me group of pyruvate secondary isotope effects allow estn. In the dimer, entrance and exit to and from the active site is slow for pyruvate and lactate, rapid for NAD.

In the tetramer, entrance and exist for pyruvate and lactate are fast while NAD exit is slow. Hydride transfer occurs at similar rates in both forms. This model explains the main features of the kinetics of both forms of the enzyme. The development of kinetic isotope effect methods for enzymic reactions has resulted in the systematic detn. Although it is early in the exptl.

The transition state for each reaction exhibits a characteristic extent of bond-breaking and bond-making, defined here as transition state poise.

Thus, concerted nucleophilic displacements SN2 or DNAN exhibit various extents of residual bond order to the leaving group and to the attacking nucleophile at the transition state.

Several concerted, sym. Enzymic transition state poise is summarized in a single diagram of bond orders using the terminol. The anal. Binding strengths of transition state analogs are readily correlated with transition state poise. Biochemistry , 42 , — , DOI: The potential energy surface is modeled by a combined quantum mech. All simulations are carried out using periodic boundary conditions at neutral pH and K.

In stage 1, a reaction coordinate is defined as the difference between the breaking and forming bond distances to the hydride ion, and a quasi-thermodn. This calcn. Then, the key interactions at the reactant, variational transition state, and product are analyzed in terms of both bond distances and electrostatic energies.

The results of both analyses support the conclusion derived from previous mutational studies that the M20 loop of DHFR makes an important contribution to the electrostatic stabilization of the hydride transfer transition state. Third, transmission coeffs. These averaged transmission coeffs. A primary kinetic isotope effect KIE of 2.

The primary KIE is mainly a consequence of the quantization of bound vibrations. In contrast, the secondary KIE, with a value of 1. American Society for Biochemistry and Molecular Biology. The kinetic expts. The isotope effects are very powerful tool for detg. Hydrostatic pressure causes a monophasic decrease in the 13C primary isotope effect expressed on the oxidn.

The primary isotope effect was measured by the competitive method, using whole-mol. Moderate pressure increases capture by activating hydride transfer, the transition state of which must therefore have a smaller vol. The decrease in the 13C isotope effect with increasing pressure means that the transition state for hydride transfer from the heavy atom must have an even smaller vol.

A similar expt. Consistent with precedence in the chem. The fact that the decrease in activation vols. The growth hormone receptor: mechanism of receptor activation, cell signaling, and physiological aspects. Front Endocrin. Maruyama IN. Activation of transmembrane cell-surface receptors via a common mechanism?

Structure and dimerization of a soluble form of B Different cell surface oligomeric states of B and B implications for signaling. Zhang X, Schwartz J. Crystal structure of the receptor-binding domain of human B insights into organization and signaling. Cytokine production and generationof CTL.

PubMed Abstract Google Scholar. Signal transduction by CD28 costimulatory receptor on T cells. B and B regulation of tyrosine kinase adaptor molecules. J Biol Chem. Role of costimulators in T cell differentiation: studies using antigen-presenting cells lacking expression of CD80 or CD Google Scholar.

T cell receptor signaling can directly enhance the avidity of CD28 ligand binding. Kinashi T. Intracellular signalling controlling integrin activation in lymphocytes. Integrin inside-out signaling and the immunological synapse. Curr Opin Cell Biol. Dependence of avidity on linker length for a bivalent ligand—bivalent receptor model system.

J Amer Chem Soc. Vauquelin G, Charlton SJ. Exploring avidity: understanding the potential gains in functional affinity and target residence time of bivalent and heterobivalent ligands. Brit J Pharmacol. The interchain disulfide linkage is not a prerequisite but enhances CD28 costimulatory function.

Cell Immunol. Understanding single-pass transmembrane receptor signaling from a structural viewpoint-what are we missing?

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Transmembrane complexes of DAP12 cystallized in lipid membranes provide insights into control of oligomerization in immunoreceptor assembly. Cell Rep. The structure of the integrin alpha-beta transmembrane complex explains integrin transmembrane signalling. EMBO J. A specific interface between integrin transmembrane helices and affinity for ligand.

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References Cherezov V, et al. Molecular control of delta-opioid receptor signalling. EMBO J. Asymmetric functioning of dimeric metabotropic glutamate receptors disclosed by positive allosteric modulators. J Biol Chem. The intracellular loops of the GB2 subunit are crucial for G-protein coupling of the heteromeric gamma-aminobutyrate B receptor. Mol Pharmacol. Evidence for a single heptahelical domain being turned on upon activation of a dimeric GPCR. Interdomain movements in metabotropic glutamate receptor activation.

Pharmacol Ther. Structural basis of glutamate recognition by a dimeric metabotropic glutamate receptor. Structural diversity of G protein-coupled receptors and significance for drug discovery. Nat Rev Drug Discov. X-ray diffraction analysis of three-dimensional crystals of bovine rhodopsin obtained from mixed micelles.

J Struct Biol. Elucidation of the role of peptide linker in calcium-sensing receptor activation process. Coupling of agonist binding to effector domain activation in metabotropic glutamate-like receptors. The cloned thrombin receptor is necessary and sufficient for activation of mitogen-activated protein kinase and mitogenesis in mouse lung fibroblasts. CFP fluorescence lifetime [ 25 , 38 ]. As shown in Fig. The decrease in the CFP-fluorescence lifetime from 2.

The control CFP-lifetime of 2. Furthermore, the experiments in Fig. The proportion of cross-linked dimers appeared to be low. Dimerization of the short form appeared to be more efficient than of the long form Fig.

However, we cannot exclude that this is due - in part - to an artefact as a result of more efficient blotting of smaller proteins. The cells were treated with the non-cell-permeable cross-linker, bis [sulfosuccinimidyl]suberate BS 3 , or left untreated, as indicated.

The positions of marker proteins that were co-electrophoresed with the samples are indicated on the left in kDa. Surprisingly, neither subtle mutations, nor deletion of the entire wedge reduced FRET significantly data not shown.

Some transmembrane domains are sufficient for dimerization, e. Averages of at least 10 individual cells from at least two independent experiments are depicted with error bars indicating the standard deviation. C Expression of the constructs that were used in B was monitored by SDS-polyacrylamide gel electrophoresis using different gels 7. Our results provide strong support for the model that dimerization is involved in regulation of RPTPs. The most reliable but less sensitive technique is FLIM.

In conclusion, ratiometric analysis of FRET by dual wavelength excitation is a sensitive method for detection of FRET, which is reliable when similar levels of the two fluorophores are expressed. BS 3 -mediated chemical cross- linking is a two-step chemical reaction, leading to a covalent bond, while FRET is only dependent on the distance between the two fluorophores. Dimerization, detected by chemical cross-linking appeared to be much less efficient than with FRET.

However, this apparent difference is caused by the relatively low efficiency of the cross-linker, BS 3. Subtle changes, for instance in the wedge, did not affect dimerization detected by FRET, but did lead to detection of reduced dimerization according to the cross-linking experiments [ 24 ]. This discrepancy may be explained by the difference in detection of dimerization. The extracellular domain may dimerize, without an interaction intracellularly.

Therefore, extracellular cross-linkers may allow detection of these dimers, while FRET analysis does not, due to the topology of the two fluorophores in the intracellular domains. For instance, cross-linking experiments demonstrated that the extracellular domain dimerized by itself [ 24 ]. This may be explained by the difference between cross-linking and FRET, as described above.

It is noteworthy that the wedge is not required for dimerization, since deletion of the wedge did not abolish dimerization Fig. Nevertheless, the wedge may be involved in stabilization of the dimer, since mutations in the wedge decreased the cross-linking efficiency [ 24 ].

Moreover, whereas the EGFR transmembrane domain by itself was not sufficient to mediate dimerization Fig. Previously, indirect detection of protein-protein interactions using chemical cross-linkers demonstrated that CD45 homodimerizes [ 44 ].

Regulation of dimerization is ill-understood. Whether Contactin functions as a ligand, or as a ligand-binding moiety remains to be determined. Whether ligand binding affects dimerization of any of these RPTPs remains to be determined. Dimerization of RPTPs may negatively regulate their activity. Mutation of a single residue in the wedge of CD45 abolished ligand-induced inactivation [ 16 ], strongly supporting the model that dimerization leads to wedge-mediated occlusion of the catalytic sites.