1-21
Bacterial transporters
Initially, carbohydrate (or sugar) molecules such as malto-oligosaccharides, sucrose, raffinose and glucose are delivered through porins (e.g. LamB, ScrY, RafY and OprB, respectively), which are located in the OM of prokaryotes (Saier, 2000a) . In the cytoplasm, these sugars are metabolized for downstream processes by proteins such as ScrB (green), ScrK (green), and RafA (yellow).
Transport of carbohydrate (or sugar) across the outer membrane of
Molecules having a molecular mass < 650 Da can passively diffuse across porins, while others are transported through specific porins (Nikaido, 1994). Sugar polymers with two (e.g. maltose and sucrose) or more units (e.g. maltodextrin) are transported by selective porins such as maltoporin (or LamB porin) (Szmelcman and Hofnung, 1975).
Transport of carbohydrate (or sugar) across the inner membrane of
Three major superfamilies, namely ABC transporters (e.g. maltose ABC transporters, PDB ID: .. 4KHZ), MFS transporters (e.g. GlpT, PDB ID: 1PW4) and PTS transporters (e.g. Glucose IICB-IIA, PDB ID: 1O2F) for carbohydrate uptake are shown in blue, orange and magenta color, respectively. Sugar Porter (SP) family 2.A.1.1 Xylose:H+ symporter Xylose Organophosphate:Pi antiporter. sn-glycerol-3-phosphate Oligosaccharide:H+ symporter. OHS) family 2.A.1.5 Lactose:H+ symporter Lactose Fucose:H+ symporter (FHS).
ATP-Binding Cassette (ABC) transporters
- ABC importer
- Carbohydrate uptake transporters family
Maltose/maltodextrin transporter from enterobacteria is a well-characterized protein member of the CUT1 family ( Oldham and Chen, 2011 ). Its domain organization is similar to that of the CUT1 family which contains a SBP, a hydrophobic TMD and an ATPase subunit (Schneider, 2001; . Koning et al., 2002).
ABC transporter architecture
- Transmembrane domains (TMDs)
- Nucleotide-binding domains (NBDs)
- Substrate (solute)-binding proteins (SBPs)
In addition, the type III importer TMD fold is associated with ABC exporters (Rees et al., 2009). During translocation, the reducing end of the sugar interacts with TM helix residues (Oldham et al., 2013).
Substrate (solute)-binding proteins (SBPs) classification
Conformational changes of the NTD (orange) and CTD (green) upon ligand binding (gray) are depicted by dotted lines. These groups possess a unique topology, where the hinge region is identified as a descriptive feature for each group (Figure 1.8).
Carbohydrate uptake mechanism via ABC importer
The conformational transition of the ABC transporter subunits SBP (green and orange), TMD (blue) and NBD (magenta) during transport of the sugar molecule (grey) is represented in the four different states 1 to 4.
IMPORTANCE OF THE STUDY
OBJECTIVES
Structural homologs of the αGlyBP protein were identified using the Dali web server ( Holm and Rosenstrom, 2010 ). The β-glycosides bound in the active site of the protein are observed to be located between the NTD and CTD.
AND METHODS………………………………… 22-39
Reagents
The Ni2+-NTA affinity resin and spiked centrifuge column used for protein purification were purchased from QIAGEN and Thermo Fisher Scientific, respectively. All crystallization buffers, plates, and other crystallization-related chemicals and tools were purchased from Hampton Research (USA) and Molecular Dimensions (UK).
Carbohydrates
For molecular cloning, restriction enzymes, ligase enzymes, and alkaline phosphatase were purchased from New England Biolabs (NEB) and/or Thermo Fisher Scientific. In addition, different kits were purchased from Himedia and QIAGEN and used for different purposes such as plasmid isolation, PCR purification and gel extraction.
METHODS
- Designing of recombinant constructs for protein overexpression
- Protein overexpression, solubilization and purification
- Protein characterization
- Crystallization of SBPs
- Data collection and processing
- Structure determination
- Model building and structure refinement
- Cross-validation
- Structure validation
- Sequence-and structure-based analysis
- Retrieval of sequence-based information
- Sequence-based analysis
- Structure-based analysis
In contrast, a homology search of the protein TTHA1301 reveals its highest similarity (sequence identity: 38% and query coverage: 96%) to an adenine-linked purine-binding protein from Brucella abortus (PDB ID: 3S99). The crystals of the protein βGlyBP_WT_FormII were obtained by incubating it with α-glycosides (melibiose, MLB and raffinose, RAF) and monosaccharides (fucose, FUC and tagatose, TAG) and high polyethylene glycol (PEG) concentration (60–70%). The PEG molecules bound to the active site of the protein βGlyBP_WT_FormII are shown as a yellow dotted sphere.
Thermodynamic parameters of β-glycosides binding to the protein βGlyBP at a physiological temperature (70°C) of the bacterium T.
IN SILICO ANALYSIS OF SBP……………………………….. 40-72
INTRODUCTION
Depending on different habitats (e.g. soil, water, animal digestive tract, etc.), microbes possess a diverse range of ABC carbohydrate transporters and metabolic pathways (Rodionov et al., 2013; Bräsen et al. , 2014). Unlike some other bacteria, it harbors only the major facilitative superfamily (MFS, a class of secondary transporters) and the ABC transporter superfamily (a class of major active transporters) for carbohydrate uptake ( Elbourne et al., 2017 ). Interestingly enough, 9 of the 11 ABC carbohydrate transport systems lack their corresponding NBD subunits ( Elbourne et al., 2017 ).
Earlier studies suggested a sharing mechanism of the NBD subunits between various carbohydrate ABC transporters to compensate for their low number (Eitinger et al., 2011).
MATERIALS AND METHODS
- Data collection
- Sequence analysis
- Structure analysis
The homologous proteins for each SBP were identified using the web tool BLAST (Altschul et al., 1990). The MSAs were further rendered using the web tool ESPript 3.0 (Gouet et al., 2003) for the clarity of the sequence alignments. The area of the active-site pocket of each SBP was calculated using the program CASTp 3.0 (Tian et al., 2018) with a default probe radius of 1.4 Å.
The interaction analysis of the linked sugar molecules to SBPs was performed using the programs Coot (Emsley et al., 2010) and PoseView (Stierand et al., 2006).
RESULTS AND DISCUSSION
- Repertoire of carbohydrate uptake ABC transporters in Thermus
- D-xylose ABC transporter
- Trehalose/Maltose ABC transporter
- Mannosylglycerate ABC transporter
- Cyclo/Maltodextrin ABC transporter
- β-glucoside transporter
- Glucose ABC transporter
- UgpABCE transporter
- Purine ABC transporter
- Sharing of nucleotide-binding domains (NBDs) and transmembrane
- Carbohydrate uptake and metabolism network in T. thermophilus HB8
The protein TTHV089 in the group of xylose-binding proteins is shown in red box. The larger docked molecules are mostly stabilized by stacking forces and few water molecules in the active site of the protein (Figure 3.4B). The active site of each protein is shown as a sphere in different colors and the rest of the protein in gray.
A sequence comparison of the protein TTH A1301 and the protein binding protein confirms the conservation of adenine-coordinating residues (Figure 3.7A).
CONCLUSION
Measurement of the binding affinity of αGlyBP for disaccharide α-glycoside proves that mutation of active-site residues changes the ligand preference. This study reports for the first time four different structural states (open-unliganded, partially-open-unliganded, open-liganded and closed-liganded) of the protein βGlyBP that reveal its conformational dynamics. All the refinement and validation statistics of the refined models are provided in Table 5.2-5.7.
Furthermore, the conserved αGlyBP/βGlyBP protein active site residues Asp118/Glu117 and Gly286/Gly297 (depend) and.
STRUCTURE OF αGlyBP……………………………………... 73-123
INTRODUCTION
Although architecturally both ABC exporters and importers contain common subunits, namely a transmembrane domain (TMD) and a nucleotide binding domain (NBD), ABC importers have an additional domain called substrate binding proteins (SBPs) (Rees et al., 2009; Wilkens , 2015). Of these, SBPs involved in carbohydrate transport belong to clusters B and D (especially subgroup D-I) (Scheepers et al., 2016). This conformational change of SBP upon ligand binding is proposed as a “Venus Fly-trap” mechanism (Mao et al., 1982).
In addition, we also found that the transport system possesses a selective mechanism based on carbohydrate length and exhibits a significant preference for disaccharides over higher oligosaccharides (Chandravanshi et al., 2019).
MATERIALS AND METHODS
- Cloning and site-directed mutagenesis
- Over expression and protein purification of wild-type and mutant
- Crystallization of wild-type (ligand bound) and mutant (ligand bound and
- Data collection, processing, structure solution, model building and
- Isothermal titration calorimetry
- Bioinformatics analysis
Data collection and refinement statistics of αGlyBP_WT protein (bound to trehalose, maltose and palatinose). Data collection and refinement statistics of αGlyBP_WT (bound to sucrose and glucose) and αGlyBP_D118A mutant protein (bound to maltose). Data collection and refinement statistics of αGlyBP_R49A (bound to maltose), αGlyBP_W287F and αGlyBP_W287A mutant proteins (bound to trehalose).
Data collection and refinement statistics for αGlyBP_W287A mutant protein (bound to maltose, palatinose and sucrose).
RESULTS
- The overall structure and the active site of αGlyBP
- αGlyBP exhibits stereo- and glycosidic-linkage selectivity
- N-terminal domain of αGlyBP dictates the open and closed conformations… 104
- Calcium ion (Ca2+) imparts the role of hinge 1 residue in conferring
- Trehalose and maltose are equally preferred by αGlyBP
- CH…π interaction is crucial for disaccharide α-glycosides binding
- α-glycosides uptake and metabolism systems are functionally associated…. 117
Overlay of glucose (GLC) bound to the active site of αGlyBP_WT•GLC with modeled (A) galactose (GAL), (B) mannose (MAN), (C) glucose-1-phosphate (G1P), (D) glucoronic acid ( GCU) and (E) arabinose (BXY). The binding affinity (Kd) of αGlyBP_R356A for disaccharide α-glycosides has been found to be in the range of. -F) Active site of αGlyBP_R356A mutant protein bound to sucrose (blue), trehalose (yellow), maltose (grey), palatinose (green) and glucose (violet), respectively.
Rather, Ca2+ of the αGlyBP_D118A•MAL complex participates in the binding and stabilization of the sugar in the active site.
CONCLUSION
The final denaturation profile of the protein βGlyBP_WT was plotted as a function of temperature and wavelength using the software Origin (version 9.6). Each flanking gene of the protein βGlyBP was manually located by analyzing the genetic context for the upstream and downstream regions. However, these carbohydrates could not be observed in the electron density map of the protein βGlyBP_WT_FormII, indicating their binding inability confirming the fluorescence and ITC data.
The overall structure of the βGlyBP protein in both forms is similar and typical of that of subgroup D-I SBPs.
STRUCTURE OF βGlyBP……………………………………… 124-183
INTRODUCTION
ABC transporters are classified into importers and exporters according to the direction (intracellular or extracellular) of the substrates transported (Wilkens, 2015; Szollosi et al., 2018). Regardless of the ligand species, SBPs have a conserved structural fold with two globular α/β domains with a central β-sheet flanked by α-helices ( Berntsson et al., 2010 ). Based on their topology, especially the hinge region, SBPs have been classified into seven different groups, A-G (Berntsson et al., 2010; Scheepers et al., 2016).
In the induced fit mechanism, an essential intermediate (open-liganded) state is known to bridge the two final (open and closed) states; it is a thermodynamically unfavorable and transient state (de Boer et al., 2019a).
MATERIALS AND METHODS
- Carbohydrates
- Construction of wild type and mutant expression plasmids
- Overexpression and protein purification of recombinant proteins
- Fluorescence spectroscopy
- Crystallization of wild type and mutant βGlyBP
- Data collection, processing and structure determination
- Measurement of ligand binding affinity using isothermal titration
- Thermal denaturation studies using circular dichroism
- Architecture of the genetic operon for β-glycosides metabolism
Using a combination of structural and thermodynamic data from the wild type and mutants, we here propose the ligand binding and selection mechanism of the protein βGlyBP. The resulting wild-type recombinant construct was further used as a template to generate the mutants of the protein βGlyBP using oligonucleotide sequences listed in Table 5.1 and Q5 Site-Directed Mutagenesis Kit (New England Biolabs, MA, USA) . All structural refinement of the model was performed using program Refmac5 (Vagin, 2004) with a default set of parameters.
After each cycle of model building, the structure was refined with the same set of parameters.
RESULTS
- Fluorescence and thermodynamic data suggest conformational
- The overall structure of the protein βGlyBP
- The protein βGlyBP exhibits a broad-range β-glycosides specificity under
- Structural basis for the ligand size selection of the protein βGlyBP
- Conserved glycosyl unit of carbohydrate renders initial ligand binding in
- Structural determinants distinguishing between the α- and β-glycosides…
- Two-step ligand-binding mechanism of the protein βGlyBP
- The C2 subdomain holds the N1 and C1 subdomains
- Second-step dynamics correlate with the differential thermodynamic
- Conformational dynamics of the N1 and C1 subdomains anchor domain
- The transport and metabolism of β-glycosides inside the cell
To investigate the former effect, circular dichroism (CD) experiment of the protein βGlyBP was performed at different temperatures ranging from 20 to 120°C. Each Glc unit of the attached β-glycosides has been labeled as Glcn (n: nth Glc number). Superimposition of the protein βGlyBP with these proteins reveals that regardless of the ligand type, all carbohydrates occupy a similar spatial position (Figure 5.10A).
Structural determinants directing carbohydrates. B) Comparison of the orientation of bound β-glycoside (CEL4, pink) to βGlyBP protein and α-glycoside (MTT, green) to MalE protein.
DISCUSSION
CONCLUSION
STRUCTURE OF U3GBP……………………………………… 184-234
INTRODUCTION
MATERIALS AND METHODS
- Preliminary in silico analysis of protein TTHA0379
- Molecular cloning for U3GBP wild type and its mutants
- Recombinant wild type and mutant protein overexpression and purification 190
- Crystallization, data collection and structure determination
- Circular dichroism measurement
- Mass spectrometry analysis
- Bioinformatics analysis of U3GBP crystal structure
RESULTS
- Protein TTHA0379 is misannotated as sugar-binding protein
