Solution structure, dynamics and binding studies of a family 11 carbohydrate-binding module from Clostridium thermocellum (CtCBM11)

2013 ◽  
Vol 451 (2) ◽  
pp. 289-300 ◽  
Author(s):  
Aldino Viegas ◽  
João Sardinha ◽  
Filipe Freire ◽  
Daniel F. Duarte ◽  
Ana L. Carvalho ◽  
...  
Keyword(s):  
Clostridium Thermocellum ◽  
Solution Structure ◽  
Close Contact ◽  
Van Der Waals Interactions ◽  
Backbone Dynamics ◽  
Titration Calorimetry ◽  
Carbohydrate Binding ◽  
Binding Studies ◽  
Carbohydrate Binding Modules ◽  
Wide Range

Non-catalytic cellulosomal CBMs (carbohydrate-binding modules) are responsible for increasing the catalytic efficiency of cellulosic enzymes by selectively putting the substrate (a wide range of poly- and oligo-saccharides) and enzyme into close contact. In the present study we carried out an atomistic rationalization of the molecular determinants of ligand specificity for a family 11 CBM from thermophilic Clostridium thermocellum [CtCBM11 (C. thermocellum CBM11)], based on a NMR and molecular modelling approach. We have determined the NMR solution structure of CtCBM11 at 25°C and 50°C and derived information on the residues of the protein that are involved in ligand recognition and on the influence of the length of the saccharide chain on binding. We obtained models of the CtCBM11–cellohexaose and CtCBM11–cellotetraose complexes by docking in accordance with the NMR experimental data. Specific ligand–protein CH-π and Van der Waals interactions were found to be determinant for the stability of the complexes and for defining specificity. Using the order parameters derived from backbone dynamics analysis in the presence and absence of ligand and at 25°C and 50°C, we determined that the protein's backbone conformational entropy is slightly positive. This data in combination with the negative binding entropy calculated from ITC (isothermal titration calorimetry) studies supports a selection mechanism where a rigid protein selects a defined oligosaccharide conformation.

scholarly journals Energetics of carbohydrate binding by a 14 kDa S-type mammalian lectin

1995 ◽  
Vol 308 (1) ◽  
pp. 237-241 ◽  
Author(s):  
R Ramkumar ◽  
A Surolia ◽  
S K Podder
Keyword(s):  
Binding Constants ◽  
Van Der Waals Interactions ◽  
Titration Calorimetry ◽  
Carbohydrate Binding ◽  
Glucose Binding ◽  
The Stability ◽  
L 14 ◽  
Derivatives Of ◽  
Binding Lectin ◽  
Site Binding

The thermodynamics of the binding of derivatives of galactose and lactose to a 14 kDa beta-galactoside-binding lectin (L-14) from sheep spleen has been studied in 10 nM phosphate/150 mM NaCl/10 mM beta-mercaptoethanol buffer, pH 7.4, and in the temperature range 285-300 K using titration calorimetry. The single-site binding constants of various sugars for the lectin were in the following order: N-acetyl-lactosamine thiodigalactoside > 4-methylumbelliferyl lactoside > lactose > 4-methylumbelliferyl alpha-D-galactoside > methyl-alpha-galactose > methyl-beta-galactose. Reactions were essentially enthalpically driven with the binding enthalpies ranging from -53.8 kJ/mol for thiodigalactoside at 301 K to -2.2 kJ/mol for galactose at 300 K, indicating that hydrogen-bonding and van der Waals interactions provide the major stabilization for these reactions. However, the binding of 4-methylumbelliferyl-alpha-D-galactose displays relatively favourable entropic contributions, indicating the existence of a non-polar site adjacent to the galactose-binding subsite. From the increments in the enthalpies for the binding of lactose, N-acetyl-lactosamine and thiodigalactoside relative to methyl-beta-galactose, the contribution of glucose binding in the subsite adjacent to that for galactose shows that glucose makes a major contribution to the stability of L-14 disaccharide complexes. Observation of enthalpy-entropy compensation for the recognition of saccharides such as lactose by L-14 and the absence of it for monosaccharides such as galactose, together with the lack of appreciable changes in the heat capacity (delta Cp), indicate that reorganization of water plays an important role in these reactions.

scholarly journals Structure-activity analysis of truncated albumin-binding domains suggests new lead constructs for potential therapeutic delivery

2020 ◽  
Vol 295 (34) ◽  
pp. 12143-12152
Author(s):  
Conan K. Wang ◽  
Anna S. Amiss ◽  
Joachim Weidmann ◽  
David J. Craik
Keyword(s):  
Structural Integrity ◽  
Cholesterol Metabolism ◽  
Activity Analysis ◽  
Titration Calorimetry ◽  
Albumin Binding ◽  
Binding Studies ◽  
Binding Domains ◽  
Structure Activity ◽  
Wide Range

Rapid clearance by renal filtration is a major impediment to the translation of small bioactive biologics into drugs. To extend serum t1/2, a commonly used approach is to attach drug leads to the G-related albumin-binding domain (ABD) to bind albumin and evade clearance. Despite the success of this approach in extending half-lives of a wide range of biologics, it is unclear whether the existing constructs are optimized for binding and size; any improvements along these lines could lead to improved drugs. Characterization of the biophysics of binding of an ABD to albumin in solution could shed light on this question. Here, we examine the binding of an ABD to human serum albumin using isothermal titration calorimetry and assess the structural integrity of the ABD using CD, NMR, and molecular dynamics. A structure-activity analysis of truncations of the ABD suggests that downsized variants could replace the full-length domain. Reducing size could have the benefit of reducing potential immunogenicity problems. We further showed that one of these variants could be used to design a bifunctional molecule with affinity for albumin and a serum protein involved in cholesterol metabolism, PCSK9, demonstrating the potential utility of these fragments in the design of cholesterol-lowering drugs. Future work could extend these in vitro binding studies to other ABD variants to develop therapeutics. Our study presents new understanding of the solution structural and binding properties of ABDs, which has implications for the design of next-generation long-lasting therapeutics.

scholarly journals Carbohydrate-binding modules influence substrate specificity of an endoglucanase from Clostridium thermocellum

2015 ◽  
Vol 80 (1) ◽  
pp. 188-192 ◽  
Author(s):  
Shunsuke Ichikawa ◽  
Mitsuki Yoshida ◽  
Shuichi Karita ◽  
Makoto Kondo ◽  
Masakazu Goto
Keyword(s):  
Substrate Specificity ◽  
Clostridium Thermocellum ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Modules

scholarly journals Carbohydrate-binding modules from a thermostable Rhodothermus marinus xylanase: cloning, expression and binding studies

2000 ◽  
Vol 345 (1) ◽  
pp. 53 ◽  
Author(s):  
Maher ABOU HACHEM ◽  
Eva NORDBERG KARLSSON ◽  
Eva BARTONEK-ROXÅ ◽  
Srinivasrao RAGHOTHAMA ◽  
Peter J. SIMPSON ◽  
...  
Keyword(s):  
Carbohydrate Binding ◽  
Rhodothermus Marinus ◽  
Binding Studies ◽  
Carbohydrate Binding Modules

Analysis of surface binding sites (SBSs) in carbohydrate active enzymes with focus on glycoside hydrolase families 13 and 77 — a mini-review

Biologia ◽  
2014 ◽  
Vol 69 (6) ◽  
Author(s):  
Darrell Cockburn ◽  
Casper Wilkens ◽  
Christian Ruzanski ◽  
Susan Andersen ◽  
Jonas Willum Nielsen ◽  
...  
Keyword(s):  
Binding Sites ◽  
Carbohydrate Binding ◽  
Surface Binding ◽  
Binding Studies ◽  
Carbohydrate Active Enzymes ◽  
Enzyme Active Site ◽  
Functional Roles ◽  
Carbohydrate Binding Modules ◽  
The Many ◽  
Key Residues

AbstractSurface binding sites (SBSs) interact with carbohydrates outside of the enzyme active site. They are frequently situated on catalytic domains and are distinct from carbohydrate binding modules (CBMs). SBSs are found in a variety of enzymes and often seen in crystal structures. Notably about half of the > 45 enzymes (in 17 GH and two GT families) with an identified SBS are from GH13 and a few from GH77, both belonging to clan GH-H of carbohydrate active enzymes. The many enzymes of GH13 with SBSs provide an opportunity to analyse their distribution within this very large and diverse family. SBS containing enzymes in GH13 are spread among 15 subfamilies (two were not assigned a subfamily). Comparison of these SBSs reveals a complex evolutionary history with evidence of conservation of key residues and/or structural location between some SBSs, while others are found at entirely distinct structural locations, suggesting convergent evolution. An array of investigations of the two SBSs in barley α-amylase demonstrated they play different functional roles in binding and degradation of polysaccharides. MalQ from Escherichia coli is an α-1,4-glucanotransferase of GH77, a family that is known to have at least one member that contains an SBS. Whereas MalQ is a single domain enzyme lacking CBMs, its plant orthologue DPE2 contains two N-terminal CBM20s. Surface plasmon resonance binding studies showed that MalQ and DPE2 have a similar affinity for β-cyclodextrin and that MalQ binds malto-oligosaccharides of >DP4 at a second site in competition with β-cyclodextrin yielding a stoichiometry >1. This suggests that MalQ may have an SBS, though its structural location remains unknown.

scholarly journals Reassembly and co-crystallization of a family 9 processive endoglucanase from its component parts: Structural and functional significance of the intermodular linker

2015 ◽  
Author(s):  
Svetlana Petkun ◽  
Inna Rozman Grinberg ◽  
Raphael Lamed ◽  
Sadanari Jindou ◽  
Tal Burstein ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Spatial Organization ◽  
Cellulase Activity ◽  
Titration Calorimetry ◽  
Carbohydrate Binding ◽  
Endoglucanase Activity ◽  
Carbohydrate Binding Modules ◽  
Processive Endoglucanase ◽  
Catalytic Module

Non-cellulosomal processive endoglucanase 9I (Cel9I) from Clostridium thermocellum is a modular protein, consisting of a family-9 glycoside hydrolase (GH9) catalytic module and two family-3 carbohydrate-binding modules (CBM3c and CBM3b), separated by linker regions. GH9 does not show cellulase activity when expressed without CBM3c and CBM3b and the presence of the CBM3c was previously shown to be essential for endoglucanase activity. Physical reassociation of independently expressed GH9 and CBM3c modules (containing linker sequences) restored 60-70% of the intact Cel9I endocellulase activity. However, the mechanism responsible for recovery of activity remained unclear. In this work we independently expressed recombinant GH9 and CBM3c with and without their interconnecting linker in Escherichia coli. We crystallized and determined the molecular structure of the GH9/linker-CBM3c heterodimer at a resolution of 1.68 Å to understand the functional and structural importance of the mutual spatial orientation of the modules and the role of the interconnecting linker during their re-association. Enzyme activity assays and isothermal titration calorimetry were performed to study and compare the effect of the linker on the re-association. The results indicated that reassembly of the modules could also occur without the linker, albeit with only very low recovery of endoglucanase activity. We propose that the linker regions in the GH9/CBM3c endoglucanases are important for spatial organization and fixation of the modules into functional enzymes.

The forkhead domain hinge-loop plays a pivotal role in DNA binding and transcriptional activity of FOXP2

2018 ◽  
Vol 399 (8) ◽  
pp. 881-893 ◽  
Author(s):  
Gavin Morris ◽  
Stoyan Stoychev ◽  
Previn Naicker ◽  
Heini W. Dirr ◽  
Sylvia Fanucchi
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Transcriptional Activity ◽  
Critical Role ◽  
Backbone Dynamics ◽  
Luciferase Reporter ◽  
Titration Calorimetry ◽  
Loop Region ◽  
Forkhead Domain ◽  
Wide Range

Abstract Forkhead box (FOX) proteins are a ubiquitously expressed family of transcription factors that regulate the development and differentiation of a wide range of tissues in animals. The FOXP subfamily members are the only known FOX proteins capable of forming domain-swapped forkhead domain (FHD) dimers. This is proposed to be due to an evolutionary mutation (P539A) that lies in the FHD hinge loop, a key region thought to fine-tune DNA sequence specificity in the FOX transcription factors. Considering the importance of the hinge loop in both the dimerisation mechanism of the FOXP FHD and its role in tuning DNA binding, a detailed investigation into the implications of mutations within this region could provide important insight into the evolution of the FOX family. Isothermal titration calorimetry and hydrogen exchange mass spectroscopy were used to study the thermodynamic binding signature and changes in backbone dynamics of FOXP2 FHD DNA binding. Dual luciferase reporter assays were performed to study the effect that the hinge-loop mutation has on FOXP2 transcriptional activity in vivo. We demonstrate that the change in dynamics of the hinge-loop region of FOXP2 alters the energetics and mechanism of DNA binding highlighting the critical role of hinge loop mutations in regulating DNA binding characteristics of the FOX proteins.

scholarly journals Carbohydrate-binding modules from a thermostable Rhodothermus marinus xylanase: cloning, expression and binding studies

1999 ◽  
Vol 345 (1) ◽  
pp. 53-60 ◽  
Author(s):  
Maher ABOU HACHEM ◽  
Eva NORDBERG KARLSSON ◽  
Eva BARTONEK-ROXÅ ◽  
Srinivasrao RAGHOTHAMA ◽  
Peter J. SIMPSON ◽  
...  
Keyword(s):  
Synergistic Effects ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Binding Affinities ◽  
Binding Assays ◽  
Rhodothermus Marinus ◽  
Binding Studies ◽  
Amorphous Cellulose ◽  
Carbohydrate Binding Modules ◽  
Cellulose Binding

The two N-terminally repeated carbohydrate-binding modules (CBM4-1 and CBM4-2) encoded by xyn10A from Rhodothermus marinus were produced in Escherichiacoli and purified by affinity chromatography. Binding assays to insoluble polysaccharides showed binding to insoluble xylan and to phosphoric-acid-swollen cellulose but not to Avicel or crystalline cellulose. Binding to insoluble substrates was significantly enhanced by the presence of Na+ and Ca2+ ions. The binding affinities for soluble polysaccharides were tested by affinity electrophoresis; strong binding occurred with different xylans and β-glucan. CBM4-2 displayed a somewhat higher binding affinity than CBM4-1 for both soluble and insoluble substrates but both had similar specificities. Binding to short oligosaccharides was measured by NMR; both modules bound with similar affinities. The binding of the modules was shown to be dominated by enthalpic forces. The binding modules did not contribute with any significant synergistic effects on xylan hydrolysis when incubated with a Xyn10A catalytic module. This is the first report of family 4 CBMs with affinity for both insoluble xylan and amorphous cellulose.

scholarly journals Reassembly and co-crystallization of a family 9 processive endoglucanase from its component parts: Structural and functional significance of the intermodular linker

2015 ◽  
Author(s):  
Svetlana Petkun ◽  
Inna Rozman Grinberg ◽  
Raphael Lamed ◽  
Sadanari Jindou ◽  
Tal Burstein ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Spatial Organization ◽  
Cellulase Activity ◽  
Titration Calorimetry ◽  
Carbohydrate Binding ◽  
Endoglucanase Activity ◽  
Carbohydrate Binding Modules ◽  
Processive Endoglucanase ◽  
Catalytic Module

Non-cellulosomal processive endoglucanase 9I (Cel9I) from Clostridium thermocellum is a modular protein, consisting of a family-9 glycoside hydrolase (GH9) catalytic module and two family-3 carbohydrate-binding modules (CBM3c and CBM3b), separated by linker regions. GH9 does not show cellulase activity when expressed without CBM3c and CBM3b and the presence of the CBM3c was previously shown to be essential for endoglucanase activity. Physical reassociation of independently expressed GH9 and CBM3c modules (containing linker sequences) restored 60-70% of the intact Cel9I endocellulase activity. However, the mechanism responsible for recovery of activity remained unclear. In this work we independently expressed recombinant GH9 and CBM3c with and without their interconnecting linker in Escherichia coli. We crystallized and determined the molecular structure of the GH9/linker-CBM3c heterodimer at a resolution of 1.68 Å to understand the functional and structural importance of the mutual spatial orientation of the modules and the role of the interconnecting linker during their re-association. Enzyme activity assays and isothermal titration calorimetry were performed to study and compare the effect of the linker on the re-association. The results indicated that reassembly of the modules could also occur without the linker, albeit with only very low recovery of endoglucanase activity. We propose that the linker regions in the GH9/CBM3c endoglucanases are important for spatial organization and fixation of the modules into functional enzymes.

scholarly journals Solution structure of family 21 carbohydrate-binding module from Rhizopus oryzae glucoamylase

2007 ◽  
Vol 403 (1) ◽  
pp. 21-30 ◽  
Author(s):  
Yu-Nan Liu ◽  
Yen-Ting Lai ◽  
Wei-I Chou ◽  
Margaret Dah-Tsyr Chang ◽  
Ping-Chiang Lyu
Keyword(s):  
Amino Acid ◽  
Chemical Shift ◽  
Ligand Binding ◽  
Solution Structure ◽  
Rhizopus Oryzae ◽  
Carbohydrate Binding ◽  
Type I ◽  
Type Ii ◽  
Sequence Motifs ◽  
Carbohydrate Binding Modules

CBMs (carbohydrate-binding modules) function independently to assist carbohydrate-active enzymes. Family 21 CBMs contain approx. 100 amino acid residues, and some members have starchbinding functions or glycogen-binding activities. We report here the first structure of a family 21 CBM from the SBD (starch-binding domain) of Rhizopus oryzae glucoamylase (RoCBM21) determined by NMR spectroscopy. This CBM has a β-sandwich fold with an immunoglobulin-like structure. Ligand-binding properties of RoCBM21 were analysed by chemical-shift perturbations and automated docking. Structural comparisons with previously reported SBDs revealed two types of topologies, namely type I and type II, with CBM20, CBM25, CBM26 and CBM41 showing type I topology, with CBM21 and CBM34 showing type II topology. According to the chemical-shift perturbations, RoCBM21 contains two ligand-binding sites. Residues in site II are similar to those found in the family 20 CBM from Aspergillus niger glucoamylase (AnCBM20). Site I, however, is embedded in a region with unique sequence motifs only found in some members of CBM21s. Additionally, docking of β-cyclodextrin and malto-oligosaccharides highlights that side chains of Y83 and W47 (one-letter amino acid code) form the central part of the conserved binding platform in the SBD. The structure of RoCBM21 provides the first direct evidence of the structural features and the basis for protein–carbohydrate recognition from an SBD of CBM21.

Sign in / Sign up

Export Citation Format

Share Document