scholarly journals Allosteric Regulation of Catalytic Activity:Escherichia coli Aspartate Transcarbamoylase versus Yeast Chorismate Mutase

2001 ◽  
Vol 65 (3) ◽  
pp. 404-421 ◽  
Author(s):  
Kerstin Helmstaedt ◽  
Sven Krappmann ◽  
Gerhard H. Braus
Keyword(s):  
Conformational Changes ◽  
Classical Model ◽  
Allosteric Regulation ◽  
Chorismate Mutase ◽  
Aspartate Transcarbamoylase ◽  
Detailed Knowledge ◽  
Wild Type ◽  
Function Relationship ◽  
Relationship Of ◽  
Entire Picture

SUMMARY Allosteric regulation of key metabolic enzymes is a fascinating field to study the structure-function relationship of induced conformational changes of proteins. In this review we compare the principles of allosteric transitions of the complex classical model aspartate transcarbamoylase (ATCase) from Escherichia coli, consisting of 12 polypeptides, and the less complicated chorismate mutase derived from baker's yeast, which functions as a homodimer. Chorismate mutase presumably represents the minimal oligomerization state of a cooperative enzyme which still can be either activated or inhibited by different heterotropic effectors. Detailed knowledge of the number of possible quaternary states and a description of molecular triggers for conformational changes of model enzymes such as ATCase and chorismate mutase shed more and more light on allostery as an important regulatory mechanism of any living cell. The comparison of wild-type and engineered mutant enzymes reveals that current textbook models for regulation do not cover the entire picture needed to describe the function of these enzymes in detail.

scholarly journals Functional studies of the rabbit intestinal Na+/glucose carrier (SGLT1) expressed in COS-7 cells: evaluation of the mutant A166C indicates this region is important for Na+-activation of the carrier

1998 ◽  
Vol 332 (1) ◽  
pp. 119-125 ◽  
Author(s):  
Steven VAYRO ◽  
Bryan LO ◽  
Mel SILVERMAN
Keyword(s):  
Glucose Transporter ◽  
Immunogold Labelling ◽  
Transport Activity ◽  
Wild Type ◽  
Functional Changes ◽  
Functional Studies ◽  
Kd Values ◽  
Function Relationship ◽  
Relationship Of ◽  
Glucose Carrier

We have exploited two mutants of the rabbit intestinal Na+/glucose carrier SGLT1 to explore the structure/function relationship of this Na+/glucose transporter in COS-7 cells. A functional N-terminal myc-epitope-tagged SGLT1 protein was constructed and used to determine the plasma-membrane localization of SGLT1. The kinetic and specificity characteristics of the myc-tagged SGLT1 mutant were identical with those of wild-type SGLT1. Immunogold labelling and electron microscopy confirmed the topology of the N-terminal region to be extracellular. Expression of the SGLT1 A166C mutant in these cells showed diminished levels of Na+-dependent α-methyl-d-glucopyranoside transport activity compared with wild-type SGLT1. For SGLT1 A166C, Vmax was 0.92±0.08 nmol/min per mg of protein and Km was 0.98±0.13 mM; for wild-type SGLT1, Vmax was 1.98±0.47 nmol/min per mg of protein and Km was 0.36±0.16 mM. Significantly, phlorrhizin (phloridzin) binding experiments confirmed equal expression of Na+-dependent high-affinity phlorrhizin binding to COS-7 cells expressing SGLT1 A166C or wild-type SGLT1 (Bmax 1.55±0.18 and 1.69±0.57 pmol/mg of protein respectively); Kd values were 0.46±0.15 and 0.51±0.11 µM for SGLT1 A166C and wild-type SGLT1 respectively. The specificity of sugar interaction was unchanged by the A166C mutation. We conclude that the replacement of an alanine residue by cysteine at position 166 has a profound effect on transporter function, resulting in a decrease in transporter turnover rate by a factor of 2. Taken as a whole the functional changes observed by SGLT1 A166C are most consistent with the mutation having caused an altered Na+ interaction with the transporter.

scholarly journals Natural Mutations Affect Structure and Function of gC1q Domain of Otolin-1

2021 ◽  
Vol 22 (16) ◽  
pp. 9085
Author(s):  
Rafał Hołubowicz ◽  
Andrzej Ożyhar ◽  
Piotr Dobryszycki
Keyword(s):  
Wild Type ◽  
Positional Vertigo ◽  
Natural Variants ◽  
Self Association ◽  
Function Relationship ◽  
The Stability ◽  
And Function ◽  
Relationship Of ◽  
Wild Type Form ◽  
Paroxysmal Positional Vertigo

Otolin-1 is a scaffold protein of otoliths and otoconia, calcium carbonate biominerals from the inner ear. It contains a gC1q domain responsible for trimerization and binding of Ca2+. Knowledge of a structure–function relationship of gC1q domain of otolin-1 is crucial for understanding the biology of balance sensing. Here, we show how natural variants alter the structure of gC1q otolin-1 and how Ca2+ are able to revert some effects of the mutations. We discovered that natural substitutions: R339S, R342W and R402P negatively affect the stability of apo-gC1q otolin-1, and that Q426R has a stabilizing effect. In the presence of Ca2+, R342W and Q426R were stabilized at higher Ca2+ concentrations than the wild-type form, and R402P was completely insensitive to Ca2+. The mutations affected the self-association of gC1q otolin-1 by inducing detrimental aggregation (R342W) or disabling the trimerization (R402P) of the protein. Our results indicate that the natural variants of gC1q otolin-1 may have a potential to cause pathological changes in otoconia and otoconial membrane, which could affect sensing of balance and increase the probability of occurrence of benign paroxysmal positional vertigo (BPPV).

scholarly journals The Glycerol-3-Phosphate Permease GlpT Is the Only Fosfomycin Transporter in Pseudomonas aeruginosa

2009 ◽  
Vol 191 (22) ◽  
pp. 6968-6974 ◽  
Author(s):  
Alfredo Castañeda-García ◽  
Alexandro Rodríguez-Rojas ◽  
Javier R. Guelfo ◽  
Jesús Blázquez
Keyword(s):  
Pseudomonas Aeruginosa ◽  
High Frequency ◽  
Wild Type ◽  
Apparent Lack ◽  
Fosfomycin Resistance ◽  
Function Relationship ◽  
Relationship Of ◽  
Specific Transport System ◽  
Insertion Mutants

ABSTRACT Fosfomycin is transported into Escherichia coli via both glycerol-3-phosphate (GlpT) and a hexose phosphate transporter (UhpT). Consequently, the inactivation of either glpT or uhpT confers increased fosfomycin resistance in this species. The inactivation of other genes, including ptsI and cyaA, also confers significant fosfomycin resistance. It has been assumed that identical mechanisms are responsible for fosfomycin transport into Pseudomonas aeruginosa cells. The study of an ordered library of insertion mutants in P. aeruginosa PA14 demonstrated that only insertions in glpT confer significant resistance. To explore the uniqueness of this resistance target in P. aeruginosa, the linkage between fosfomycin resistance and the use of glycerol-3-phosphate was tested. Fosfomycin-resistant (Fos-R) mutants were obtained in LB and minimal medium containing glycerol as the sole carbon source at a frequency of 10−6. However, no Fos-R mutants grew on plates containing fosfomycin and glycerol-3-phosphate instead of glycerol (mutant frequency, ≤5 × 10−11). In addition, 10 out of 10 independent spontaneous Fos-R mutants, obtained on LB-fosfomycin, harbored mutations in glpT, and in all cases the sensitivity to fosfomycin was recovered upon complementation with the wild-type glpT gene. The analysis of these mutants provides additional insights into the structure-function relationship of glycerol-3-phosphate the transporter in P. aeruginosa. Studies with glucose-6-phosphate and different mutant derivatives strongly suggest that P. aeruginosa lacks a specific transport system for this sugar. Thus, glpT seems to be the only fosfomycin resistance mutational target in P. aeruginosa. The high frequency of Fos-R mutations and their apparent lack of fitness cost suggest that Fos-R variants will be obtained easily in vivo upon the fosfomycin treatment of P. aeruginosa infections.

scholarly journals Structure-function relationship of serine protease-protein inhibitor interaction.

2001 ◽  
Vol 48 (2) ◽  
pp. 419-428 ◽  
Author(s):  
J Otlewski ◽  
M Jaskólski ◽  
O Buczek ◽  
T Cierpicki ◽  
H Czapińska ◽  
...  
Keyword(s):  
High Resolution ◽  
Structure Function ◽  
Serine Proteases ◽  
Cathepsin G ◽  
Wild Type ◽  
Stability Parameters ◽  
X Ray ◽  
Structure Function Relationship ◽  
Function Relationship ◽  
Relationship Of

We report our progress in understanding the structure-function relationship of the interaction between protein inhibitors and several serine proteases. Recently, we have determined high resolution solution structures of two inhibitors Apis mellifera chymotrypsin inhibitor-1 (AMCI-I) and Linum usitatissimum trypsin inhibitor (LUTI) in the free state and an ultra high resolution X-ray structure of BPTI. All three inhibitors, despite totally different scaffolds, contain a solvent exposed loop of similar conformation which is highly complementary to the enzyme active site. Isothermal calo- rimetry data show that the interaction between wild type BPTI and chymotrypsin is entropy driven and that the enthalpy component opposes complex formation. Our research is focused on extensive mutagenesis of the four positions from the protease binding loop of BPTI: P1, P1', P3, and P4. We mutated these residues to different amino acids and the variants were characterized by determination of the association constants, stability parameters and crystal structures of protease-inhibitor complexes. Accommodation of the P1 residue in the S1 pocket of four proteases: chymotrypsin, trypsin, neutrophil elastase and cathepsin G was probed with 18 P1 variants. High resolution X-ray structures of ten complexes between bovine trypsin and P1 variants of BPTI have been determined and compared with the cognate P1 Lys side chain. Mutations of the wild type Ala16 (P1') to larger side chains always caused a drop of the association constant. According to the crystal structure of the Leu16 BPTI-trypsin complex, introduction of the larger residue at the P1' position leads to steric conflicts in the vicinity of the mutation. Finally, mutations at the P4 site allowed an improvement of the association with several serine proteases involved in blood clotting. Conversely, introduction of Ser, Val, and Phe in place of Gly12 (P4) had invariably a destabilizing effect on the complex with these proteases.

scholarly journals Crystal structure of jumping spider rhodopsin-1 as a light sensitive GPCR

2019 ◽  
Vol 116 (29) ◽  
pp. 14547-14556 ◽  
Author(s):  
Niranjan Varma ◽  
Eshita Mutt ◽  
Jonas Mühle ◽  
Valérie Panneels ◽  
Akihisa Terakita ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Conformational Changes ◽  
Jumping Spider ◽  
Downstream Signaling ◽  
Class A ◽  
Bovine Rhodopsin ◽  
Function Relationship ◽  
Relationship Of ◽  
G Protein Coupled

Light-sensitive G protein-coupled receptors (GPCRs)—rhodopsins—absorb photons to isomerize their covalently bound retinal, triggering conformational changes that result in downstream signaling cascades. Monostable rhodopsins release retinal upon isomerization as opposed to the retinal in bistable rhodopsins that “reisomerize” upon absorption of a second photon. Understanding the mechanistic differences between these light-sensitive GPCRs has been hindered by the scarcity of recombinant models of the latter. Here, we reveal the high-resolution crystal structure of a recombinant bistable rhodopsin, jumping spider rhodopsin-1, bound to the inverse agonist 9-cis retinal. We observe a water-mediated network around the ligand hinting toward the basis of their bistable nature. In contrast to bovine rhodopsin (monostable), the transmembrane bundle of jumping spider rhodopsin-1 as well that of the bistable squid rhodopsin adopts a more “activation-ready” conformation often observed in other nonphotosensitive class A GPCRs. These similarities suggest the role of jumping spider rhodopsin-1 as a potential model system in the study of the structure–function relationship of both photosensitive and nonphotosensitive class A GPCRs.

scholarly journals Folding–function relationship of the most common cystic fibrosis–causing CFTR conductance mutants

2019 ◽  
Vol 2 (1) ◽  
pp. e201800172 ◽  
Author(s):  
Marcel van Willigen ◽  
Annelotte M Vonk ◽  
Hui Ying Yeoh ◽  
Evelien Kruisselbrink ◽  
Bertrand Kleizen ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Class Iii ◽  
Wild Type ◽  
Limited Function ◽  
Disease Variant ◽  
Function Relationship ◽  
Therapeutic Compounds ◽  
Relationship Of ◽  
Class Iv ◽  
Novel Therapeutic

Cystic fibrosis is caused by mutations in the CFTR gene, which are subdivided into six classes. Mutants of classes III and IV reach the cell surface but have limited function. Most class-III and class-IV mutants respond well to the recently approved potentiator VX-770, which opens the channel. We here revisited function and folding of some class-IV mutants and discovered that R347P is the only one that leads to major defects in folding. By this criterion and by its functional response to corrector drug VX-809, R347P qualifies also as a class-II mutation. Other class-IV mutants folded like wild-type CFTR and responded similarly to VX-809, demonstrating how function and folding are connected. Studies on both types of defects complement each other in understanding how compounds improve mutant CFTR function. This provides an attractive unbiased approach for characterizing mode of action of novel therapeutic compounds and helps address which drugs are efficacious for each cystic fibrosis disease variant.

scholarly journals Uranyl Binding to Proteins and Structural-Functional Impacts

Biomolecules ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 457 ◽  
Author(s):  
Ying-Wu Lin
Keyword(s):  
Conformational Changes ◽  
Protein Interactions ◽  
Metal Binding ◽  
Structural Information ◽  
Metal Binding Sites ◽  
Biological Remediation ◽  
Protein Dna Interactions ◽  
Ligand Interactions ◽  
Function Relationship ◽  
Relationship Of

The widespread use of uranium for civilian purposes causes a worldwide concern of its threat to human health due to the long-lived radioactivity of uranium and the high toxicity of uranyl ion (UO22+). Although uranyl–protein/DNA interactions have been known for decades, fewer advances are made in understanding their structural-functional impacts. Instead of focusing only on the structural information, this article aims to review the recent advances in understanding the binding of uranyl to proteins in either potential, native, or artificial metal-binding sites, and the structural-functional impacts of uranyl–protein interactions, such as inducing conformational changes and disrupting protein-protein/DNA/ligand interactions. Photo-induced protein/DNA cleavages, as well as other impacts, are also highlighted. These advances shed light on the structure-function relationship of proteins, especially for metalloproteins, as impacted by uranyl–protein interactions. It is desired to seek approaches for biological remediation of uranyl ions, and ultimately make a full use of the double-edged sword of uranium.

scholarly journals Structure-function relationship of Gossypium hirsutum NAC transcription factor, GhNAC4 with regard to ABA and abiotic stress responses

2020 ◽  
Author(s):  
Vikas Shalibhadra Trishla ◽  
Pulugurtha Bharadwaja Kirti
Keyword(s):  
Transcription Factor ◽  
Abiotic Stress ◽  
Stress Tolerance ◽  
Abiotic Stress Tolerance ◽  
Nac Transcription Factor ◽  
Wild Type ◽  
Nac Domain ◽  
Transcriptional Regulatory ◽  
Function Relationship ◽  
Relationship Of

AbstractOur previous study demonstrated that the expression of GhNAC4, a NAC transcription factor from cotton, was induced by abiotic stresses and abscisic acid (ABA) treatment. In the present study, we investigated the molecular mechanisms underlying ABA and stress response of GhNAC4. Over-expression of GhNAC4 in transgenic tobacco conferred tolerance to salinity and drought treatments with associated enhanced expression of several stress-responsive marker genes. GhNAC4 is a nuclear protein that exhibits transcriptional activation property, and the C-terminal transcriptional regulatory (TR) domain is responsible for it. GhNAC4 also forms homo-dimers and the N-terminal NAC-domain is essential for this activity. The structure-function relationship of NAC transcription factors, particularly with respect to abiotic stress tolerance remains largely unclear. In this study, we investigated the domains essential for the biochemical functions of GhNAC4. We developed transgenic tobacco plants overexpressing the GhNAC4 NAC-domain and the TR-domain separately. NAC-domain transgenics showed hypersensitivity to exogenous ABA while TR-domain transgenics exhibited reduced sensitivity. Abiotic stress assays indicated that transgenic plants expressing both the domains separately were more tolerant than wild-type plants with the NAC-domain transgenics showing increased tolerance as compared to TR-domain transgenics. Expression analysis revealed that various stress-responsive genes were upregulated in both NAC-domain and TR-domain transgenics as compared to wild-type under salinity and drought treatments. These results suggest that the stress tolerance ability of GhNAC4 is associated with both the component domains while the ABA responsiveness is largely associated with N-terminal NAC-domain.Key MessageNAC and transcriptional regulatory domains are responsible for the abiotic stress tolerance ability of the cotton NAC transcription factor GhNAC4 while the ABA-responsiveness is largely associated with the NAC-domain.

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