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Molecular analysis of cyclic ?-maltosyl-(1?6)-maltose binding protein in the bacterial metabolic pathway.

ABSTRACT: Cyclic ?-maltosyl-(1?6)-maltose (CMM) is a cyclic glucotetrasaccharide with alternating ?-1,4 and ?-1,6 linkages. Here, we report functional and structural analyses on CMM-binding protein (CMMBP), which is a substrate-binding protein (SBP) of an ABC importer system of the bacteria Arthrobacter globiformis. Isothermal titration calorimetry analysis revealed that CMMBP specifically bound to CMM with a Kd value of 9.6 nM. The crystal structure of CMMBP was determined at a resolution of 1.47 Å, and a panose molecule was bound in a cleft between two domains. To delineate its structural features, the crystal structure of CMMBP was compared with other SBPs specific for carbohydrates, such as cyclic ?-nigerosyl-(1?6)-nigerose and cyclodextrins. These results indicate that A. globiformis has a unique metabolic pathway specialized for CMM.

SUBMITTER: Kohno M

PROVIDER: S-EPMC7676653 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Molecular analysis of cyclic α-maltosyl-(1→6)-maltose binding protein in the bacterial metabolic pathway.

Kohno Masaki M Arakawa Takatoshi T Sunagawa Naoki N Mori Tetsuya T Igarashi Kiyohiko K Nishimoto Tomoyuki T Fushinobu Shinya S

PloS one 20201119 11

Cyclic α-maltosyl-(1→6)-maltose (CMM) is a cyclic glucotetrasaccharide with alternating α-1,4 and α-1,6 linkages. Here, we report functional and structural analyses on CMM-binding protein (CMMBP), which is a substrate-binding protein (SBP) of an ABC importer system of the bacteria Arthrobacter globiformis. Isothermal titration calorimetry analysis revealed that CMMBP specifically bound to CMM with a Kd value of 9.6 nM. The crystal structure of CMMBP was determined at a resolution of 1.47 Å, and ...[more]

PMID: 33211750

Similar Datasets

Structural features of a bacterial cyclic α-maltosyl-(1→6)-maltose (CMM) hydrolase critical for CMM recognition and hydrolysis.

Project description:Cyclic α-maltosyl-(1→6)-maltose (CMM, cyclo-{→6)-α-d-Glcp-(1→4)-α-d-Glcp-(1→6)-α-d-Glcp-(1→4)-α-d-Glcp-(1→})is a cyclic glucotetrasaccharide with alternating α-1,4 and α-1,6 linkages. CMM is composed of two maltose units and is one of the smallest cyclic glucooligosaccharides. Although CMM is resistant to usual amylases, it is efficiently hydrolyzed by CMM hydrolase (CMMase), belonging to subfamily 20 of glycoside hydrolase family 13 (GH13_20). Here, we determined the ligand-free crystal structure of CMMase from the soil-associated bacterium Arthrobacter globiformis and its structures in complex with maltose, panose, and CMM to elucidate the structural basis of substrate recognition by CMMase. The structures disclosed that although the monomer structure consists of three domains commonly adopted by GH13 and other α-amylase-related enzymes, CMMase forms a unique wing-like dimer structure. The complex structure with CMM revealed four specific subsites, namely -3', -2, -1, and +1'. We also observed that the bound CMM molecule adopts a low-energy conformer compared with the X-ray structure of a single CMM crystal, also determined here. Comparison of the CMMase active site with those in other enzymes of the GH13_20 family revealed that three regions forming the wall of the cleft, denoted PYF (Pro-203/Tyr-204/Phe-205), CS (Cys-163/Ser-164), and Y (Tyr-168), are present only in CMMase and are involved in CMM recognition. Combinations of multiple substitutions in these regions markedly decreased the activity toward CMM, indicating that the specificity for this cyclic tetrasaccharide is supported by the entire shape of the pocket. In summary, our work uncovers the mechanistic basis for the highly specific interactions of CMMase with its substrate CMM.

| S-EPMC6204909 | biostudies-literature

Structural insight into how Streptomyces coelicolor maltosyl transferase GlgE binds α-maltose 1-phosphate and forms a maltosyl-enzyme intermediate.

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Stimulation of the maltose transporter ATPase by unliganded maltose binding protein.

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Accessing a hidden conformation of the maltose binding protein using accelerated molecular dynamics.

Project description:Periplasmic binding proteins (PBPs) are a large family of molecular transporters that play a key role in nutrient uptake and chemotaxis in Gram-negative bacteria. All PBPs have characteristic two-domain architecture with a central interdomain ligand-binding cleft. Upon binding to their respective ligands, PBPs undergo a large conformational change that effectively closes the binding cleft. This conformational change is traditionally viewed as a ligand induced-fit process; however, the intrinsic dynamics of the protein may also be crucial for ligand recognition. Recent NMR paramagnetic relaxation enhancement (PRE) experiments have shown that the maltose binding protein (MBP) - a prototypical member of the PBP superfamily - exists in a rapidly exchanging (ns to µs regime) mixture comprising an open state (approx 95%), and a minor partially closed state (approx 5%). Here we describe accelerated MD simulations that provide a detailed picture of the transition between the open and partially closed states, and confirm the existence of a dynamical equilibrium between these two states in apo MBP. We find that a flexible part of the protein called the balancing interface motif (residues 175-184) is displaced during the transformation. Continuum electrostatic calculations indicate that the repacking of non-polar residues near the hinge region plays an important role in driving the conformational change. Oscillations between open and partially closed states create variations in the shape and size of the binding site. The study provides a detailed description of the conformational space available to ligand-free MBP, and has implications for understanding ligand recognition and allostery in related proteins.

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Peracetylated alpha-D-glucopyranosyl fluoride and peracetylated alpha-maltosyl fluoride.

Project description:The X-ray analyses of 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranosyl fluoride, C(14)H(19)FO(9), (I), and the corresponding maltose derivative 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranosyl-(1-->4)-2,3,6-tri-O-acetyl-alpha-D-glucopyranosyl fluoride, C(26)H(35)FO(17), (II), are reported. These add to the series of published alpha-glycosyl halide structures; those of the peracetylated alpha-glucosyl chloride [James & Hall (1969). Acta Cryst. A25, S196] and bromide [Takai, Watanabe, Hayashi & Watanabe (1976). Bull. Fac. Eng. Hokkaido Univ. 79, 101-109] have been reported already. In our structures, which have been determined at 140 K, the glycopyranosyl ring appears in a regular (4)C(1) chair conformation with all the substituents, except for the anomeric fluoride (which adopts an axial orientation), in equatorial positions. The observed bond lengths are consistent with a strong anomeric effect, viz. the C1-O5 (carbohydrate numbering) bond lengths are 1.381 (2) and 1.381 (3) A in (I) and (II), respectively, both significantly shorter than the C5-O5 bond lengths, viz. 1.448 (2) A in (I) and 1.444 (3) A in (II).

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Conformational Dynamics in the Binding-Protein-Independent Mutant of the Escherichia coli Maltose Transporter, MalG511, and Its Interaction with Maltose Binding Protein.

Project description:MalG511 is a genetically selected binding-protein-independent mutant of the Escherichia coli maltose transporter MalFGK2, which retains specificity for maltose and shows a high basal ATPase activity in the absence of maltose binding protein (MBP). It shows an intriguing biphasic behavior in maltose transport assays in the presence of MBP, with low levels of MBP stimulating the activity and higher levels (>50 μM) inhibiting the transport activity. Remarkably, the rescuing effect of the MBP suppressor mutant, MBPG13D, turns it into an attractive model for studying regulatory mechanisms in the ABC transporter superfamily. It is hypothesized that the special characteristics of MalG511 result from mutations that shift its equilibrium toward the transition state of MalFGK2. We tested this hypothesis by using site-directed spin labeling in combination with electron paramagnetic resonance spectroscopy, which showed conformational changes in MalG511 and its interaction with MBP and MBPG13D during its catalytic cycle. We found that MalG511 utilizes the same alternate access mechanism as MalFGK2, including all three open, semi-open, and closed states of the MalK dimer, to transport maltose across the membrane. However, the equilibrium of this mutant is shifted toward the semi-open state in its resting state and interacts with MBP with high affinity, providing an explanation for the inhibition of MalG511 by MBP at higher concentrations. In contrast, the mutant binding protein, MBPG13D, interacts with lower affinity and could restore MalG511 to a normal catalytic cycle.

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Full engagement of liganded maltose-binding protein stabilizes a semi-open ATP-binding cassette dimer in the maltose transporter.

Project description:MalFGK2 is an ATP-binding cassette (ABC) transporter that mediates the uptake of maltose/maltodextrins into Escherichia coli. A periplasmic maltose-binding protein (MBP) delivers maltose to the transmembrane subunits (MalFG) and stimulates the ATPase activity of the cytoplasmic nucleotide-binding subunits (MalK dimer). This MBP-stimulated ATPase activity is independent of maltose for purified transporter in detergent micelles. However, when the transporter is reconstituted in membrane bilayers, only the liganded form of MBP efficiently stimulates its activity. To investigate the mechanism of maltose stimulation, electron paramagnetic resonance spectroscopy was used to study the interactions between the transporter and MBP in nanodiscs and in detergent. We found that full engagement of both lobes of maltose-bound MBP unto MalFGK2 is facilitated by nucleotides and stabilizes a semi-open MalK dimer. Maltose-bound MBP promotes the transition to the semi-open state of MalK when the transporter is in the membrane, whereas such regulation does not require maltose in detergent. We suggest that stabilization of the semi-open MalK2 conformation by maltose-bound MBP is key to the coupling of maltose transport to ATP hydrolysis in vivo, because it facilitates the progression of the MalK dimer from the open to the semi-open conformation, from which it can proceed to hydrolyze ATP.

| S-EPMC4715599 | biostudies-literature

The energetics of structural change in maltose-binding protein.

Project description: Not available

| S-EPMC240646 | biostudies-literature

Folding of maltose binding protein outside of and in GroEL.

Project description:We used hydrogen exchange-mass spectrometry (HX MS) and fluorescence to compare the folding of maltose binding protein (MBP) in free solution and in the GroEL/ES cavity. Upon refolding, MBP initially collapses into a dynamic molten globule-like ensemble, then forms an obligatory on-pathway native-like folding intermediate (1.2 seconds) that brings together sequentially remote segments and then folds globally after a long delay (30 seconds). A single valine to glycine mutation imposes a definable folding defect, slows early intermediate formation by 20-fold, and therefore subsequent global folding by approximately twofold. Simple encapsulation within GroEL repairs the folding defect and reestablishes fast folding, with or without ATP-driven cycling. Further examination exposes the structural mechanism. The early folding intermediate is stabilized by an organized cluster of 24 hydrophobic side chains. The cluster preexists in the collapsed ensemble before the H-bond formation seen by HX MS. The V9G mutation slows folding by disrupting the preintermediate cluster. GroEL restores wild-type folding rates by restabilizing the preintermediate, perhaps by a nonspecific equilibrium compression effect within its tightly confining central cavity. These results reveal an active GroEL function other than previously proposed mechanisms, suggesting that GroEL possesses different functionalities that are able to relieve different folding problems. The discovery of the preintermediate, its mutational destabilization, and its restoration by GroEL encapsulation was made possible by the measurement of a previously unexpected type of low-level HX protection, apparently not dependent on H-bonding, that may be characteristic of proteins in confined spaces.

| S-EPMC5776991 | biostudies-literature

Dynamics of alpha-helical subdomain rotation in the intact maltose ATP-binding cassette transporter.

Project description:ATP-binding cassette (ABC) transporters are powered by a nucleotide-binding domain dimer that opens and closes during cycles of ATP hydrolysis. These domains consist of a RecA-like subdomain and an α-helical subdomain that is specific to the family. Many studies on isolated domains suggest that the helical subdomain rotates toward the RecA-like subdomain in response to ATP binding, moving the family signature motif into a favorable position to interact with the nucleotide across the dimer interface. Moreover, the transmembrane domains are docked into a cleft at the interface between these subdomains, suggesting a putative role of the rotation in interdomain communication. Electron paramagnetic resonance spectroscopy was used to study the dynamics of this rotation in the intact Escherichia coli maltose transporter MalFGK(2). This importer requires a periplasmic maltose-binding protein (MBP) that activates ATP hydrolysis by promoting the closure of the cassette dimer (MalK(2)). Whereas this rotation occurred during the transport cycle, it required not only trinucleotide, but also MBP, suggesting it is part of a global conformational change in the transporter. Interaction of AMP-PNP-Mg(2+) and a MBP that is locked in a closed conformation induced a transition from open MalK(2) to semiopen MalK(2) without significant subdomain rotation. Inward rotation of the helical subdomain and complete closure of MalK(2) therefore appear to be coupled to the reorientation of transmembrane helices and the opening of MBP, events that promote transfer of maltose into the transporter. After ATP hydrolysis, the helical subdomain rotates out as MalK(2) opens, resetting the transporter in an inward-facing conformation.

| S-EPMC2996640 | biostudies-literature

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