Structural determinants of allosteric regulation in alternatively spliced AMPA receptors

Abstract Structure-function studies contribute to deciphering how small modifications in the primary structure could introduce desirable characteristics into enzymes without affecting its overall functioning. Malic enzymes (ME) are ubiquitous and responsible for a wide variety of functions. The availability of a high number of ME crystal structures from different species facilitates comparisons between sequence and structure. Specifically, the structural determinants necessary for fumarate allosteric regulation of ME has been of particular interest. NADP-ME2 from Arabidopsis thaliana exhibits a distinctive and complex regulation by fumarate, acting as an activator or an inhibitor according to substrate and effector concentrations. However, the 3D structure for this enzyme is not yet reported. In this work, we characterized the NADP-ME2 allosteric site by structural modeling, molecular docking, normal mode analysis and mutagenesis. The regulatory site model and its docking analysis suggested that other C4 acids including malate, NADP-ME2 substrate, could also fit into fumarate’s pocket. Besides, a non-conserved cluster of hydrophobic residues in the second sphere of the allosteric site was identified. The substitution of one of those residues, L62, by a less flexible residue as tryptophan, resulted in a complete loss of fumarate activation and a reduction of substrate affinities for the active site. In addition, normal mode analysis indicated that conformational changes leading to the activation could originate in the region surrounding L62, extending through the allosteric site till the active site. Finally, the results in this work contribute to the understanding of structural determinants necessary for allosteric regulation providing new insights for enzyme optimization.

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Structural Determinants for High-Affinity Binding of Insulin-Like Growth Factor II to Insulin Receptor (IR)-A, the Exon 11 Minus Isoform of the IR

Molecular Endocrinology ◽

10.1210/me.2004-0183 ◽

2004 ◽

Vol 18 (10) ◽

pp. 2502-2512 ◽

Cited By ~ 127

Author(s):

Adam Denley ◽

Eric R. Bonython ◽

Grant W. Booker ◽

Leah J. Cosgrove ◽

Briony E. Forbes ◽

...

Keyword(s):

Insulin Receptor ◽

Cell Types ◽

Positive Control ◽

Affinity Binding ◽

Structural Determinants ◽

High Affinity ◽

Igf I ◽

High Affinity Receptor ◽

Alternatively Spliced ◽

Exon 11

Abstract The insulin receptor (IR) lacking the alternatively spliced exon 11 (IR-A) is preferentially expressed in fetal and cancer cells. The IR-A has been identified as a high-affinity receptor for insulin and IGF-II but not IGF-I, which it binds with substantially lower affinity. Several cancer cell types that express the IR-A also overexpress IGF-II, suggesting a possible autocrine proliferative loop. To determine the regions of IGF-I and IGF-II responsible for this differential affinity, chimeras were made where the C and D domains were exchanged between IGF-I and IGF-II either singly or together. The abilities of these chimeras to bind to, and activate, the IR-A were investigated. We also investigated the ability of these chimeras to bind and activate the IR exon 11+ isoform (IR-B) and as a positive control, the IGF-I receptor (IGF-1R). We show that the C domain and, to a lesser extent, the D domains represent the principal determinants of the binding differences between IGF-I and IGF-II to IR-A. The C and D domains of IGF-II promote higher affinity binding to the IR-A than the equivalent domains of IGF-I, resulting in an affinity close to that of insulin for the IR-A. The C and D domains also regulate the IR-B binding specificity of the IGFs in a similar manner, although the level of binding for all IGF ligands to IR-B is lower than to IR-A. In contrast, the C and D domains of IGF-I allow higher affinity binding to the IGF-1R than the analogous domains of IGF-II. Activation of IGF-1R by the chimeras reflected their binding affinities whereas the phosphorylation of the two IR isoforms was more complex.

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Nanoscale Mobility of the Apo State and TARP Stoichiometry Dictate the Gating Behavior of Alternatively Spliced AMPA Receptors

Neuron ◽

10.1016/j.neuron.2019.03.046 ◽

2019 ◽

Vol 102 (5) ◽

pp. 976-992.e5 ◽

Cited By ~ 9

Author(s):

G. Brent Dawe ◽

Md. Fahim Kadir ◽

Raminta Venskutonytė ◽

Amanda M. Perozzo ◽

Yuhao Yan ◽

...

Keyword(s):

Ampa Receptors ◽

Alternatively Spliced ◽

Apo State

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Allosteric Regulation of the Photosynthetic C4 Isoenzyme of Phosphoenolpyruvate Carboxylase: A Comparative Study Between Enzymes from Monocot and Dicot Plants

Journal of the Mexican Chemical Society ◽

10.29356/jmcs.v56i1.275 ◽

2017 ◽

Vol 56 (1) ◽

Author(s):

Rodrigo Güémez-Toro ◽

Carlos Mújica-Jiménez ◽

Rosario A. Muñoz-Clares

Keyword(s):

Phosphoenolpyruvate Carboxylase ◽

Allosteric Regulation ◽

Three Dimensional ◽

Dimensional Structure ◽

Velocity Data ◽

Structural Determinants ◽

Multiple Alignments ◽

High Flexibility ◽

Loop Conformation ◽

Conserved Residue

In the present study, we have investigated the complex allosteric regulation of the non-phosphorylated forms of the photosynthetic phosphoenolpyruvate carboxylase isoenzymes (PEPC-C4) from amaranth (AhPEPC-C4) and maize (ZmPEPC-C4) leaves. Previous studies showed that glycine (Gly) only activates PEPC-C4 from monocot plants, as maize, but not from dicot plants, as amaranth. Our initial velocity data confirm this, in spite that AhPEPC-C4 binds Gly with much higher affinity than ZmPEPC-C4. In AhPEPC-C4, the lack of Gly activation is overcome mainly by its higher affinity for the substrate phosphoenolpyruvate and its lower affinity for the inhibitor malate compared with ZmPEPC-C4. We have also explored the structural determinants of the differences in Gly activation by performing multiple alignments between the known monocot and dicot PEPC-C4 sequences and by modeling, in both the AhPEPC-C4 and ZmPEPC-C4 isoenzymes, the three-dimensional structure of the loop proposed as the Gly binding site, which was not observed in the crystal structure of the maize enzyme due to its high flexibility. The models suggest that conserved lysyl and aspartyl residues are important for binding to the activator molecule, and that a nearby non-conserved residue may be responsible for differences between the amaranth and maize enzymes in the loop conformation, which would account for the poorer affinity for Gly of the maize enzyme as well as for its higher degree of activation.

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