Protein Linkers Provide Limits on the Domain Interactions in the ABC Importer GlnPQ and Determine the Rate of Transport

GlnPQ is an ATP-binding cassette importer with a unique domain organization and intricate transport behavior. The protein has two extracytoplamic substrate-binding domains (SBDs) per membrane subunit, each with different specificity for amino acids and different spacing to the translocator domain. We determined the effect of the length and structure of the linkers, which connect the SBDs to each other and to the membrane-embedded translocator domain, on the transport by GlnPQ. We reveal that varying the linker length impacts transport in a dual manner that depends on the conformational dynamics of the SBD. Varying the linker length not only changes the time for the SBD to find the translocator (docking) but also changes the probability to release the substrate again, thus altering the transport efficiency. On the basis of the experimental data and mathematical modeling, we calculate the docking efficiency as function of linker length and lifetime of the closed conformation. Importantly, not only linker length but also features in the sequence are important for efficient delivery of substrate from SBD to the translocator. We show that the linkers provide a platform for SBD docking and are not merely flexible structures.     

Direct interaction of eag domains and cyclic nucleotide–binding homology domains regulate deactivation gating in hERG channels

Human ether-á-go-go (eag)-related gene (hERG) potassium channels play a critical role in cardiac repolarization and are characterized by unusually slow closing (deactivation) kinetics. The N-terminal “eag” domain and a C-terminal C-linker/cyclic nucleotide–binding homology domain (CNBHD) are required for regulation of slow deactivation. The region between the S4 and S5 transmembrane domains (S4–S5 linker) is also implicated in this process, but the mechanism for regulation of slow deactivation is unclear. Here, using an eag domain–deleted channel (hERG Δeag) fused to Citrine fluorescent protein, we found that most channels bearing individual alanine mutations in the S4–S5 linker were directly regulated by recombinant eag domains fused to a cyan fluorescent protein (N-eag-CFP) and had robust Förster resonance energy transfer (FRET). Additionally, a channel bearing a group of eight alanine residues in the S4–S5 linker was not measurably regulated by N-eag-CFP domains, but robust FRET was measured. These findings demonstrate that the eag domain associated with all of the S4–S5 linker mutant channels. In contrast, channels that also lacked the CNBHD (hERG Δeag ΔCNBHD-Citrine) were not measurably regulated by N-eag-CFP nor was FRET detected, suggesting that the C-linker/CNBHD was required for eag domains to directly associate with the channel. In a FRET hybridization assay, N-eag-CFP had robust FRET with a C-linker/CNBHD-Citrine, suggesting a direct and specific interaction between the eag domain and the C-linker/CNBHD. Lastly, coexpression of a hERG subunit lacking the CNBHD and the distal C-terminal region (hERG ΔpCT-Citrine) with hERG Δeag-CFP subunits had FRET and partial restoration of slow deactivation. Collectively, these findings reveal that the C-linker/CNBHD, but not the S4–S5 linker, was necessary for the eag domain to associate with the channel, that the eag domain and the C-linker/CNBHD were sufficient for a direct interaction, and that an intersubunit interaction between the eag domain and the C-linker/CNBHD regulated slow deactivation in hERG channels at the plasma membrane.     

Energy Coupling Efficiency in the Type I ABC Transporter GlnPQ

Solute transport via ATP binding cassette (ABC) importers involves receptor-mediated substrate binding, which is followed by ATP-driven translocation of the substrate across the membrane. How these steps are exactly initiated and coupled, and how much ATP it takes to complete a full transport cycle, are subject of debate. Here, we reconstitute the ABC importer GlnPQ in nanodiscs and in proteoliposomes and determine substrate-(in)dependent ATP hydrolysis and transmembrane transport. We determined the conformational states of the substrate-binding domains (SBDs) by single-molecule Förster resonance energy transfer measurements. We find that the basal ATPase activity (ATP hydrolysis in the absence of substrate) is mainly caused by the docking of the closed-unliganded state of the SBDs onto the transporter domain of GlnPQ and that, unlike glutamine, arginine binds both SBDs but does not trigger their closing. Furthermore, comparison of the ATPase activity in nanodiscs with glutamine transport in proteoliposomes shows that the stoichiometry of ATP per substrate is close to two. These findings help understand the mechanism of transport and the energy coupling efficiency in ABC transporters with covalently linked SBDs, which may aid our understanding of Type I ABC importers in general.     

Crystal structures of the starch-binding domain from Rhizopus oryzae glucoamylase reveal a polysaccharide-binding path

GA (glucoamylase) hydrolyses starch and polysaccharides to beta-D-glucose. RoGA (Rhizopus oryzae GA) consists of two functional domains, an N-terminal SBD (starch-binding domain) and a C-terminal catalytic domain, which are connected by an O-glycosylated linker. In the present study, the crystal structures of the SBD from RoGA (RoGACBM21) and the complexes with beta-cyclodextrin (SBD-betaCD) and maltoheptaose (SBD-G7) were determined. Two carbohydrate binding sites, I (Trp(47)) and II (Tyr(32)), were resolved and their binding was co-operative. Besides the hydrophobic interaction, two unique polyN loops comprising consecutive asparagine residues also participate in the sugar binding. A conformational change in Tyr(32) was observed between unliganded and liganded SBDs. To elucidate the mechanism of polysaccharide binding, a number of mutants were constructed and characterized by a quantitative binding isotherm and Scatchard analysis. A possible binding path for long-chain polysaccharides in RoGACBM21 was proposed.     

A single residue mutation abolishes attachment of the CBM26 starch-binding domain from <Emphasis Type="Italic">Lactobacillus amylovorus</Emphasis> α-amylase

Starch is degraded by amylases that frequently have a modular structure composed of a catalytic domain and at least one non-catalytic domain that is involved in polysaccharide binding. The C-terminal domain from the Lactobacillus amylovorus α-amylase has an unusual architecture composed of five tandem starch-binding domains (SBDs). These domains belong to family 26 in the carbohydrate-binding modules (CBM) classification. It has been reported that members of this family have only one site for starch binding, where aromatic amino acids perform the binding function. In SBDs, fold similarities are better conserved than sequences; nevertheless, it is possible to identify in CBM26 members at least two aromatic residues highly conserved. We attempt to explain polysaccharide recognition for the L. amylovorus α–amylase SBD through site-directed mutagenesis of aromatic amino acids. Three amino acids were identified as essential for binding, two tyrosines and one tryptophan. Y18L and Y20L mutations were found to decrease the SBD binding capacity, but unexpectedly, the mutation at W32L led to a total loss of affinity, either with linear or ramified substrates. The critical role of Trp 32 in substrate binding confirms the presence of just one binding site in each α-amylase SBD.     

The nucleotide‐bound/substrate‐bound conformation of the Mycoplasma genitalium DnaK chaperone

Hsp70 chaperones keep protein homeostasis facilitating the response of organisms to changes in external and internal conditions. Hsp70s have two domains—nucleotide binding domain (NBD) and substrate binding domain (SBD)—connected by a conserved hydrophobic linker. Functioning of Hsp70s depend on tightly regulated cycles of ATP hydrolysis allosterically coupled, often together with cochaperones, to the binding/release of peptide substrates. Here we describe the crystal structure of the Mycoplasma genitalium DnaK (MgDnaK) protein, an Hsp70 homolog, in the noncompact, nucleotide‐bound/substrate‐bound conformation. The MgDnaK structure resembles the one from the thermophilic eubacteria DnaK trapped in the same state. However, in MgDnaK the NBD and SBD domains remain close to each other despite the lack of direct interaction between them and with the linker contacting the two subdomains of SBD. These observations suggest that the structures might represent an intermediate of the protein where the conserved linker binds to the SBD to favor the noncompact state of the protein by stabilizing the SBDβ‐SBDα subdomains interaction, promoting the capacity of the protein to sample different conformations, which is critical for proper functioning of the molecular chaperone allosteric mechanism. Comparison of the solved structures indicates that the NBD remains essentially invariant in presence or absence of nucleotide.     

Role of the N-terminal starch-binding domains in the kinetic properties of starch synthase III from Arabidopsis thaliana

Starch synthase III (SSIII), one of the SS isoforms involved in plant starch synthesis, has been reported to play a regulatory role in the synthesis of transient starch. SSIII from Arabidopsis thaliana contains 1025 amino acid residues and has an N-terminal transit peptide for chloroplast localization which is followed by three repeated starch-binding domains (SBDs; SSIII residues 22-591) and a C-terminal catalytic domain (residues 592-1025) similar to bacterial glycogen synthase. In this work, we constructed recombinant full-length and truncated isoforms of SSIII, lacking one, two, or three SBDs, and recombinant proteins, containing three, two, or one SBD, to investigate the role of these domains in enzyme activity. Results revealed that SSIII uses preferentially ADPGlc, although UDPGlc can also be used as a sugar donor substrate. When ADPGlc was used, the presence of the SBDs confers particular properties to each isoform, increasing the apparent affinity and the V max for the oligosaccharide acceptor substrate. However, no substantial changes in the kinetic parameters for glycogen were observed when UDPGlc was the donor substrate. Under glycogen saturating conditions, the presence of SBDs increases progressively the apparent affinity and V max for ADPGlc but not for UDPGlc. Adsorption assays showed that the N-terminal region of SSIII, containing three, two, or one SBD module have increased capacity to bind starch depending on the number of SBD modules, with the D23 protein (containing the second and third SBD module) being the one that makes the greatest contribution to binding. The results presented here suggest that the N-terminal SBDs have a regulatory role, showing a starch binding capacity and modulating the catalytic properties of SSIII.     

On the role of the two extracytoplasmic substrate-binding domains in the ABC transporter OpuA

Members of two transporter families of the ATP-binding cassette (ABC) superfamily use two or even four extracytoplasmic substrate-binding domains (SBDs) for transport. We report on the role of the two SBDs in the translocation cycle of the ABC transporter OpuA from Lactococcus lactis. Heterooligomeric OpuA complexes with only one SBD or one functional and one non-functional SBD (inactivated by covalent linkage of a substrate mimic) have been constructed, and the substrate binding and transport kinetics of the purified transporters, reconstituted in liposomes, have been determined. The data indicate that the two SBDs of OpuA interact in a cooperative manner in the translocation process by stimulating either the docking of the SBDs onto the translocator or the delivery of glycine betaine to the translocator. It appears that one of these initial steps, but not the later steps in translocation or resetting of the system to the initial state, is rate determining for transport. These new insights on the functional role of the extracytoplasmic SBDs are discussed in the light of the current knowledge of substrate-binding-protein-dependent ABC transporters.     

Amide hydrogen exchange reveals conformational changes in hsp70 chaperones important for allosteric regulation

Hsp70 chaperones assist protein folding processes by a nucleotide-driven cycle of substrate binding and release. Although structural information is available for the isolated nucleotide-binding (NBD) and substrate-binding domains (SBD) in the high affinity conformation, the low affinity conformations and the conformational changes associated with mutual allosteric regulation remained largely enigmatic. By using amide hydrogen exchange in combination with mass spectrometry, we analyzed the Escherichia coli Hsp70 homologue DnaK as full-length protein and its individual domains in the nucleotide-free and ATP-bound conformation. We found a surprising degree of flexibility in both domains. The comparison of the full-length protein with the isolated domains demonstrates a mutual stabilization of both domains. This protection from solvent was most pronounced and in addition was nucleotide-dependent in the lowerbeta-sheet of the SBD and the loop that connects the last beta-strand with helix alphaA. Interestingly, the linker region, which connects NBD and SBD and which is close to the protected loop in the SBD, is solvent-exposed in the absence of nucleotide and completely protected from hydrogen exchange in the presence of ATP. Peptide binding to DnaK.ATP reverts the ATP-induced conformational changes in the linker and selected parts of the NBD. Our data outline a pathway for allosteric interdomain control and suggest an important role of the linker and the base of helix alphaA.     

Cloning and Characterization of the Polyhydroxybutyrate Depolymerase Gene of Pseudomonas stutzeri and Analysis of the Function of Substrate-Binding Domains

The extracellular polyhydroxybutyrate (PHB) depolymerase gene (phaZ_Pst) of Pseudomonas stutzeri was cloned and sequenced. phaZ_Pst was composed of 1,728 bp encoding a protein of 576 amino acids. Analyses of the N-terminal amino acid sequence and the matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) mass spectrum of the purified enzyme showed that the mature enzyme consisted of 538 amino acids with a deduced molecular mass of 57,506 Da. Analysis of the deduced amino acid sequence of the protein revealed a domain structure containing a catalytic domain, putative linker region, and two putative substrate-binding domains (SBDI and SBDII). The putative linker region was similar to the repeating units of the cadherin-like domain of chitinase A from Vibrio harveyi and chitinase B from Clostridium paraputrificum. The binding characteristics of SBDs to poly([R]-3-hydroxybutyrate) [P(3HB)] and chitin granules were characterized by using fusion proteins of SBDs with glutathione S-transferase (GST). These GST fusion proteins with SBDII and SBDI showed binding activity toward P(3HB) granules but did not bind on chitin granules. It has been suggested that the SBDs of the depolymerase interact specifically with the surface of P(3HB). In addition, a kinetic analysis for the enzymatic hydrolysis of 3-hydroxybutyrate oligomers of various sizes has suggested that the catalytic domain of the enzyme recognizes at least two monomeric units as substrates.     

Cyclic nucleotide selectivity of protein kinase G isozymes

The intrinsic activity of the C‐terminal catalytic (C) domain of cyclic guanosine monophosphate (cGMP)‐dependent protein kinases (PKG) is inhibited by interactions with the N‐terminal regulatory (R) domain. Selective binding of cGMP to cyclic nucleotide binding (CNB) domains within the R‐domain disrupts the inhibitory R–C interaction, leading to the release and activation of the C‐domain. Affinity measurements of mammalian and plasmodium PKG CNB domains reveal different degrees of cyclic nucleotide affinity and selectivity; the CNB domains adjacent to the C‐domain are more cGMP selective and therefore critical for cGMP‐dependent activation. Crystal structures of isolated CNB domains in the presence and absence of cyclic nucleotides reveal isozyme‐specific contacts that explain cyclic nucleotide selectivity and conformational changes that accompany CNB. Crystal structures of tandem CNB domains identify two types of CNB‐mediated dimeric contacts that indicate cGMP‐driven reorganization of domain–domain interfaces that include large conformational changes. Here, we review the available structural and functional information of PKG CNB domains that further advance our understanding of cGMP mediated regulation and activation of PKG isozymes.     

Alpha-Amylase Starch Binding Domains: Cooperative Effects of Binding to Starch Granules of Multiple Tandemly Arranged Domains

The Lactobacillus amylovorus alpha-amylase starch binding domain (SBD) is a functional domain responsible for binding to insoluble starch. Structurally, this domain is dissimilar from other reported SBDs because it is composed of five identical tandem modules of 91 amino acids each. To understand adsorption phenomena specific to this SBD, the importance of their modular arrangement in relationship to binding ability was investigated. Peptides corresponding to one, two, three, four, or five modules were expressed as His-tagged proteins. Protein binding assays showed an increased capacity of adsorption as a function of the number of modules, suggesting that each unit of the SBD may act in an additive or synergic way to optimize binding to raw starch.     

Alpha-amylase starch binding domains: cooperative effects of binding to starch granules of multiple tandemly arranged domains

The Lactobacillus amylovorus alpha-amylase starch binding domain (SBD) is a functional domain responsible for binding to insoluble starch. Structurally, this domain is dissimilar from other reported SBDs because it is composed of five identical tandem modules of 91 amino acids each. To understand adsorption phenomena specific to this SBD, the importance of their modular arrangement in relationship to binding ability was investigated. Peptides corresponding to one, two, three, four, or five modules were expressed as His-tagged proteins. Protein binding assays showed an increased capacity of adsorption as a function of the number of modules, suggesting that each unit of the SBD may act in an additive or synergic way to optimize binding to raw starch.     

Structural insights into the functional versatility of WW domain-containing oxidoreductase tumor suppressor

Recent work on WW domain-containing oxidoreductase (WWOX) tumor suppressor is beginning to shed new light on both the molecular mechanism of action of its WW domains as well as the contiguous catalytic domain. Herein, the structural basis underlying the ability of WW1 domain to bind to various physiological ligands and how the orphan WW2 tandem partner synergizes its ligand binding in the context of WW1–WW2 tandem module of WWOX is discussed. Notably, the WW domains within the WW1–WW2 tandem module physically associate so as to adopt a fixed spatial orientation relative to each other. In this manner, the association of WW2 domain with WW1 hinders ligand binding to the latter. Consequently, ligand binding to WW1 domain not only results in the displacement of WW2 lid but also disrupts the fixed orientation of WW domains in the liganded conformation. Equally importantly, structure-guided functional approach suggests that the catalytic domain of WWOX likely serves as a retinal oxidoreductase that catalyzes the reversible oxidation and reduction of all-trans-retinal. Collectively, this review provides structural insights into the functional versatility of a key signaling protein with important implications on its biology.     

Starch-binding domain shuffling in Aspergillus niger glucoamylase

Aspergillus niger glucoamylase (GA) consists mainly of two forms, GAI [from the N-terminus, catalytic domain + linker + starch-binding domain (SBD)] and GAII (catalytic domain + linker). These domains were shuffled to make RGAI (SBD + linker + catalytic domain), RGAIDeltaL (SBD + catalytic domain) and RGAII (linker + catalytic domain), with domains defined by function rather than by tertiary structure. In addition, Paenibacillus macerans cyclomaltodextrin glucanotransferase SBD replaced the closely related A.niger GA SBD to give GAE. Soluble starch hydrolysis rates decreased as RGAII approximately GAII approximately GAI > RGAIDeltaL approximately RGAI approximately GAE. Insoluble starch hydrolysis rates were GAI > RGAIDeltaL > RGAI > GAE approximately RGAII > GAII, while insoluble starch-binding capacities were GAI > RGAI > RGAIDeltaL > RGAII > GAII > GAE. These results indicate that: (i) moving the SBD to the N-terminus or replacing the native SBD somewhat affects soluble starch hydrolysis; (ii) SBD location significantly affects insoluble starch binding and hydrolysis; (iii) insoluble starch hydrolysis is imperfectly correlated with its binding by the SBD; and (iv) placing the P.macerans cyclomaltodextrin glucanotransferase SBD at the end of a linker, instead of closely associated with the rest of the enzyme, severely reduces its ability to bind and hydrolyze insoluble starch.     

Structural basis of J cochaperone binding and regulation of Hsp70

The many protein processing reactions of the ATP-hydrolyzing Hsp70s are regulated by J cochaperones, which contain J domains that stimulate Hsp70 ATPase activity and accessory domains that present protein substrates to Hsp70s. We report the structure of a J domain complexed with a J responsive portion of a mammalian Hsp70. The J domain activates ATPase activity by directing the linker that connects the Hsp70 nucleotide binding domain (NBD) and substrate binding domain (SBD) toward a hydrophobic patch on the NBD surface. Binding of the J domain to Hsp70 displaces the SBD from the NBD, which may allow the SBD flexibility to capture diverse substrates. Unlike prokaryotic Hsp70, the SBD and NBD of the mammalian chaperone interact in the ADP state. Thus, although both nucleotides and J cochaperones modulate Hsp70 NBD:linker and NBD:SBD interactions, the intrinsic persistence of those interactions differs in different Hsp70s and this may optimize their activities for different cellular roles.     

N-terminal and Central Segments of the Type 1 Ryanodine Receptor Mediate Its Interaction with FK506-binding Proteins

We used site-directed labeling of the type 1 ryanodine receptor (RyR1) and fluorescence resonance energy transfer (FRET) measurements to map RyR1 sequence elements forming the binding site of the 12-kDa binding protein for the immunosuppressant drug, FK506. This protein, FKBP12, promotes the RyR1 closed state, thereby inhibiting Ca²⁺ leakage in resting muscle. Although FKBP12 function is well established, its binding determinants within the RyR1 protein sequence remain unresolved. To identify these sequence determinants using FRET, we created five single-Cys FKBP variants labeled with Alexa Fluor 488 (denoted D-FKBP) and then targeted these D-FKBPs to full-length RyR1 constructs containing decahistidine (His₁₀) “tags” placed within N-terminal (amino acid residues 76–619) or central (residues 2157–2777) regions of RyR1. The FRET acceptor Cy3NTA bound specifically and saturably to these His tags, allowing distance analysis of FRET measured from each D-FKBP variant to Cy3NTA bound to each His tag. Results indicate that D-FKBP binds proximal to both N-terminal and central domains of RyR1, thus suggesting that the FKBP binding site is composed of determinants from both regions. These findings further imply that the RyR1 N-terminal and central domains are proximal to one another, a core premise of the domain-switch hypothesis of RyR function. We observed FRET from GFP fused at position 620 within the N-terminal domain to central domain His-tagged sites, thus further supporting this hypothesis. Taken together, these results support the conclusion that N-terminal and central domain elements are closely apposed near the FKBP binding site within the RyR1 three-dimensional structure.     

A Highlights from MBoC Selection: Role of the loop L4,5 in allosteric regulation in mtHsp70s: in vivo significance of domain communication and its implications in protein translocation

Mitochondrial Hsp70 (mtHsp70) is essential for a vast repertoire of functions, including protein import, and requires effective interdomain communication for efficient partner-protein interactions. However, the in vivo functional significance of allosteric regulation in eukaryotes is poorly defined. Using integrated biochemical and yeast genetic approaches, we provide compelling evidence that a conserved substrate-binding domain (SBD) loop, L_4,5, plays a critical role in allosteric communication governing mtHsp70 chaperone functions across species. In yeast, a temperature-sensitive L_4,5 mutation (E467A) disrupts bidirectional domain communication, leading to compromised protein import and mitochondrial function. Loop L_4,5 functions synergistically with the linker in modulating the allosteric interface and conformational transitions between SBD and the nucleotide-binding domain (NBD), thus regulating interdomain communication. Second-site intragenic suppressors of E467A isolated within the SBD suppress domain communication defects by conformationally altering the allosteric interface, thereby restoring import and growth phenotypes. Strikingly, the suppressor mutations highlight that restoration of communication from NBD to SBD alone is the minimum essential requirement for effective in vivo function when primed at higher basal ATPase activity, mimicking the J-protein–bound state. Together these findings provide the first mechanistic insights into critical regions within the SBD of mtHsp70s regulating interdomain communication, thus highlighting its importance in protein translocation and mitochondrial biogenesis.     

Cloning, expression and characterization of a poly(3-hydroxybutyrate) depolymerase from Marinobacter sp. NK-1

A DNA fragment carrying the gene encoding poly(3-hydroxybutyrate) (P(3HB)) depolymerase was cloned from the genomic DNA of Marinobacter sp. DNA sequencing analysis revealed that the Marinobacter sp. P(3HB) depolymerase gene is composed of 1734 bp and encodes 578 amino acids with a molecular mass of 61,757 Da. A sequence homology search showed that the deduced protein contains the signal peptide, catalytic domain (CD), cadherin-type linker domain (LD), and two substrate-binding domain (SBD). The fusion proteins of glutathione S-transferase (GST) with the CD showed the hydrolytic activity for denatured P(3HB) (dP(3HB)), P(3HB) emulsion (eP(3HB)) and p-nitrophenylbutyrate. On the other hand, the fusion proteins lacking the SBD showed much lower hydrolytic activity for dP(3HB) compared to the proteins containing both CD and SBD. In addition, binding tests revealed that the SBDs are specifically bound not to eP(3HB) but dP(3HB). These suggest that the SBDs play a crucial role in the enzymatic hydrolysis of dP(3HB) that is a solid substrate.     

The crystal structure of the CmABCB1 G132V mutant,which favors the outward‐facing state,reveals the mechanism of the pivotal joint between TM1 and TM3

CmABCB1 is a homologue of human P‐glycoprotein, which extrudes various substrates by iterative cycles of conformational changes between the inward‐ and outward‐facing states. Comparison of the inward‐ and outward‐facing structures of CmABCB1 suggested that pivotal joints in the transmembrane domain regulate the tilt of transmembrane helices. Transmembrane helix 1 (TM1) forms a tight helix–helix contact with TM3 at the TM1–3 joint. Mutation of Gly132 to valine at the TM1–3 joint, G132V, caused a 10‐fold increase in ATPase activity, but the mechanism underlying this change remains unclear. Here, we report a crystal structure of the outward‐facing state of the CmABCB1 G132V mutant at a 2.15 Å resolution. We observed structural displacements between the outward‐facing states of G132V and the previous one at the region around the TM1–3 joint, and a significant expansion at the extracellular gate. We hypothesize that steric hindrance caused by the Val substitution shifted the conformational equilibrium toward the outward‐facing state, favoring the dimeric state of the nucleotide‐binding domains and thereby increasing the ATPase activity of the G132V mutant.