Substrate-binding Domain Research Articles

The structure of monoamine oxidases: past, present, and future.

The first crystal structure of mammalian monoamine oxidases (MAOs) was solved in 2002; almost 65years after, these FAD-dependent enzymes were discovered and classified as responsible for the oxidation of aromatic neurotransmitters. Both MAO A and MAO B feature a two-domain topology characterized by the Rossmann fold, interacting with dinucleotide cofactors, which is intimately associated to a substrate-binding domain. This globular body is endowed with a C-terminal α-helix that anchors the protein to the outer mitochondrial phospholipid bilayer. As monotopic membrane proteins, the structural elucidation of MAOs was a challenging task that required the screening of different detergent conditions for their purification and crystallization. MAO A and MAO B structures differ both in their oligomerization architecture and in details of their active sites. Purified human MAO B and rat MAO A are dimeric, whereas human MAO A was found to be monomeric, which is believed to result from the detergent treatments used to extract the protein from the membrane. The active site of MAOs consists of a hydrophobic cavity located in front of the flavin cofactor and extending to the protein surface. Some structural features are highly conserved in the two isozymes, such as a Tyr-Tyr aromatic sandwich in front of the flavin ring and a Lys residue hydrogen-bonded to the cofactor N5 atom, whereas a pair of gating residues (Phe208/Ile335 in MAO A; Ile199/Tyr326 in MAO B) specifically determines the different substrate and inhibitor properties of the two enzymes.

Human glucose-regulated protein 78 modulates intracellular production and secretion of nonstructural protein 1 of dengue virus.

Virus-host interactions play important roles in virus infection and host cellular response. Several viruses, including dengue virus (DENV), usurp host chaperones to support their amplification and survival in the host cell. We investigated the interaction of nonstructural protein 1 (NS1) of DENV with three endoplasmic reticulum-resident chaperones (i.e. GRP78, calnexin and calreticulin) to delineate their functional roles and potential binding sites for protein complex formation. GRP78 protein showed prominent association with DENV NS1 in virus-infected Huh7 cells as evidenced by co-localization and co-immunoprecipitation assays. Further studies on the functional interaction of GRP78 protein were performed by using siRNA-mediated gene knockdown in a DENV replicon transfection system. GRP78 knockdown significantly decreased intracellular NS1 production and delayed NS1 secretion but had no effect on viral RNA replication. Dissecting the important domain of GRP78 required for DENV NS1 interaction showed co-immunoprecipitation of DENV NS1 with a full-length and substrate-binding domain (SBD), but not an ATPase domain, of GRP78, confirming their interaction through SBD binding. Molecular dynamics simulations of DENV NS1 and human GRP78 complex revealed their potential binding sites through hydrogen and hydrophobic bonding. The majority of GRP78-binding sites were located in a β-roll domain and connector subdomains on the DENV NS1 structure involved in hydrophobic surface formation. Taken together, our findings demonstrated the roles of human GRP78 in facilitating the intracellular production and secretion of DENV NS1 as well as predicted potential binding sites between the DENV NS1 and GRP78 complex, which could have implications in the future development of target-based antiviral drugs.

Mechanical properties of BiP protein determined by nano‐rheology

Immunoglobulin Binding Protein (BiP) is a chaperone and molecular motor belonging to the Hsp70 family, involved in the regulation of important biological processes such as synthesis, folding and translocation of proteins in the Endoplasmic Reticulum. BiP has two highly conserved domains: the N-terminal Nucleotide-Binding Domain (NBD), and the C-terminal Substrate-Binding Domain (SBD), connected by a hydrophobic linker. ATP binds and it is hydrolyzed to ADP in the NBD, and BiP's extended polypeptide substrates bind in the SBD. Like many molecular motors, BiP function depends on both structural and catalytic properties that may contribute to its performance. One novel approach to study the mechanical properties of BiP considers exploring the changes in the viscoelastic behavior upon ligand binding, using a technique called nano-rheology. This technique is essentially a traditional rheology experiment, in which an oscillatory force is directly applied to the protein under study, and the resulting average deformation is measured. Our results show that the folded state of the protein behaves like a viscoelastic material, getting softer when it binds nucleotides- ATP, ADP, and AMP-PNP-, but stiffer when binding HTFPAVL peptide substrate. Also, we observed that peptide binding dramatically increases the affinity for ADP, decreasing it dissociation constant (KD ) around 1000 times, demonstrating allosteric coupling between SBD and NBD domains.

Characterization of a novel potential peptide import system in Treponema denticola

Treponema denticola is a major etiologic agent of chronic periodontitis. On the outer sheath of T. denticola, several proteins, such as the major outer sheath protein and dentilisin were detected, and among them, a 95 kDa protein which has not yet been characterized. The aim of this study was to characterize the function of this 95 kDa protein containing gene cluster. A gene encoding this 95 kDa protein (TDE_1072) of T. denticola was inactivated by homologous recombination. We compared growth curves between the TDE_1072 mutant and wild-type strains as well as differences in gene expression by DNA microarray analysis. Differential expression of genes identified by microarray analysis was confirmed by quantitative reverse transcription-polymerase chain reaction. The proteins encoded by TDE_1072, TDE_1073, TDE_1074, TDE_1075, and TDE_1076 shared respective similarities to the substrate-binding domain (DppA) of an ABC-type dipeptide/oligopeptide/nickel transport system, and to the permease components (DppB and DppC) and ATPase components (DppD and DppF) of an ABC-type dipeptide/oligopeptide/nickel transport system. Inactivation of dppA attenuated the growth of T. denticola and dppA-dppF were co-transcribed. In contrast, expression of oppB-oppF was up-regulated in the mutant. Our findings indicate that TDE_1072 may be a potential periplasmic solute binding protein encoded by dppA that is involved in the organization of a peptide uptake system with dppB-dppF.

Inward-facing conformation of l-ascorbate transporter suggests an elevator mechanism

Various bacteria can ferment vitamin C (l-ascorbate) under anaerobic conditions via the phosphoenolpyruvate-dependent phosphotransferase system (PTS). The PTSasc system is composed of two soluble energy-coupling proteins (EI and HPr) and an enzyme II complex (EIIA, EIIB, and EIIC) for the anaerobic uptake of ascorbate and its phosphorylation to l-ascorbate 6-phosphate in vivo. Crystal structures of the ascorbate-bound EIIC component from Escherichia coli are available in outward-open and occluded conformations, suggesting a possible elevator mechanism of membrane transport. Despite these advances, it remains unclear how EIIC actually transports the substrate across the membrane and interacts with EIIB, which transfers its phosphate group to the EIIC-embedding ascorbate. Here, we present the crystal structure of the EIICasc component from Pasteurella multocida in the inward-facing conformation. By comparing three conformational states, we confirmed the original proposed model: the ascorbate translocation can be achieved by a rigid-body movement of the substrate-binding core domain relative to the V motif domain, which brings along the transmembrane helices TM2 and TM7 of the V motif domain to undergo a winding at the pivotal positions. Together with an in vivo transport assay, we completed the picture of the transport cycle of the ascorbate superfamily of membrane-spanning EIIC components of the PTS system.

The disorderly conduct of Hsc70 and its interaction with the Alzheimer's-related Tau protein

Hsp70 chaperones bind to various protein substrates for folding, trafficking, and degradation. Considerable structural information is available about how prokaryotic Hsp70 (DnaK) binds substrates, but less is known about mammalian Hsp70s, of which there are 13 isoforms encoded in the human genome. Here, we report the interaction between the human Hsp70 isoform heat shock cognate 71-kDa protein (Hsc70 or HSPA8) and peptides derived from the microtubule-associated protein Tau, which is linked to Alzheimer's disease. For structural studies, we used an Hsc70 construct (called BETA) comprising the substrate-binding domain but lacking the lid. Importantly, we found that truncating the lid does not significantly impair Hsc70's chaperone activity or allostery in vitro Using NMR, we show that BETA is partially dynamically disordered in the absence of substrate and that binding of the Tau sequence GKVQIINKKG (with a KD = 500 nm) causes dramatic rigidification of BETA. NOE distance measurements revealed that Tau binds to the canonical substrate-binding cleft, similar to the binding observed with DnaK. To further develop BETA as a tool for studying Hsc70 interactions, we also measured BETA binding in NMR and fluorescent competition assays to peptides derived from huntingtin, insulin, a second Tau-recognition sequence, and a KFERQ-like sequence linked to chaperone-mediated autophagy. We found that the insulin C-peptide binds BETA with high affinity (KD < 100 nm), whereas the others do not (KD > 100 μm). Together, our findings reveal several similarities and differences in how prokaryotic and mammalian Hsp70 isoforms interact with different substrate peptides.

Heterodimeric O-methyltransferases involved in the biosynthesis of noscapine in opium poppy.

Noscapine biosynthesis in opium poppy involves three characterized O-methyltransferases (OMTs) and a fourth responsible for the 4'-methoxyl on the phthalide isoquinoline scaffold. The first three enzymes are homodimers, whereas the latter is a heterodimer encoded by two linked genes (OMT2 and OMT3). Neither OMT2 nor OMT3 form stable homodimers, but yield a substrate-specific heterodimer when their genes are co-expressed in Escherichia coli. The only substrate, 4'-O-desmethyl-3-O-acetylpapaveroxine, is a seco-berbine pathway intermediate that undergoes ester hydrolysis subsequent to 4'-O-methylation leading to the formation of narcotine hemiacetal. In the absence of 4'-O-methylation, a parallel pathway yields narcotoline hemiacetal. Dehydrogenation produces noscapine and narcotoline from the corresponding hemiacetals. Phthalide isoquinoline intermediates with a 4'-hydroxyl (i.e. narcotoline and narcotoline hemiacetal), or the corresponding 1-hydroxyl on protoberberine intermediates, were not accepted. Norcoclaurine 6OMT, which shares 81% amino acid sequence identity with OMT3, also formed a functionally similar heterodimer with OMT2. Suppression of OMT2 transcript levels in opium poppy increased narcotoline accumulation, whereas reduced OMT3 transcript abundance caused no detectable change in the alkaloid phenotype. Opium poppy chemotype Marianne accumulates high levels of narcotoline and showed no detectable OMT2:OMT3 activity. Compared with the active subunit from the Bea's Choice chemotype, Marianne OMT2 exhibited a single S122Y mutation in the dimerization domain that precluded heterodimer formation based on homology models. Both subunits contributed to the formation of the substrate-binding domain, although site-directed mutagenesis revealed OMT2 as the active subunit. The occurrence of physiologically relevant OMT heterodimers increases the catalytic diversity of enzymes derived from a smaller number of gene products.

Heterologous expression and enhanced production of β-1,4-glucanase of Bacillus halodurans C-125 in Escherichia coli

Abstract Background Recombinant DNA technology enables us to produce proteins with desired properties and insubstantial amount for industrial applications. Endo-1, 4-β-glucanases (Egl) is one of the major enzyme involved in degradation of cellulose, an important component of plant cell wall. The present study was aimed at enhancing the production of endo-1, 4-β-glucanases (Egl) of Bacillus halodurans in Escherichia coli. Results A putative Egl gene of Bacillus Halodurans was expressed in E. coli by cloning in pET 22b (+). On induction with isopropyl-b- d -1-thiogalactopyranoside, the enzyme expression reached upto ~ 20% of the cell protein producing 29.2 mg/liter culture. An increase in cell density to 12 in auto-inducing LB medium (absorbance at 600 nm) enhanced β-glucanase production up to 5.4 fold. The molecular mass of the enzyme was determined to be 39 KDa, which is nearly the same as the calculated value. Protein sequence was analyzed by CDD, Pfam, I TASSER, COACH, PROCHECK Servers and putative amino acids involved in the formation of catalytic, substrate and metal binding domains were identified. Phylogenetic analysis of the β-glucanases of B. halodurans was performed and position of Egl among other members of the genus Bacillus producing endo-glucanases was determined. Temperature and pH optima of the enzyme were found to be 60°C and 8.0, respectively, under the assay conditions. Conclusion Production of endo-1, 4 β-glucanase enzymes from B. halodurans increased several folds when cloned in pET vector and expressed in E. coli. To our knowledge, this is the first report of high-level expression and characterization of an endo-1, 4 β-glucanases from B. halodurans. How to cite: Zeeshan N, Naz S, Naz S et al. Heterologous expression and enhanced production of β-1,4-glucanase of Bacillus halodurans C-125 in E. coli. Electron J Biotechnol 2018;34. https://doi.org/10.1016/j.ejbt.2018.05.001 .

Intra-molecular pathways of allosteric control in Hsp70s.

The 70 kDa heat-shock protein (Hsp70) is undoubtedly the most versatile of all molecular chaperones. Hsp70 is involved in numerous cellular protein folding processes, accompanying proteins throughout their lifespan from de novo folding at the ribosome to degradation at the proteasome, surveilling protein stability and functionality. Several properties of this ATP-dependent chaperone constitute the molecular basis for this versatility. With its substrate binding domain (SBD), Hsp70 transiently interacts with a short degenerative linear sequence motif found practically in all proteins and, in addition, with more folded protein conformers. Binding to polypeptides is tightly regulated by ATP binding and hydrolysis in the nucleotide binding domain, which is coupled to the SBD by an intricate allosteric mechanism. Hsp70 is regulated by a host of J-cochaperones, which act as targeting factors by regulating the ATPase activity of Hsp70 in synergism with the substrates themselves, and by several families of nucleotide exchange factors. In this review, I focus on the allosteric mechanism, which allows Hsp70s to interact with substrates with ultrahigh affinity through a non-equilibrium mode of action and summarize what mutagenesis and structural studies have taught us about the pathways and mechanics of interdomain communication.This article is part of a discussion meeting issue 'Allostery and molecular machines'.

1262 HSP70iQ435A-encoding DNA repigments vitiligo lesions in Sinclair swine

HSP70iQ435A carries an amino acid modification within the substrate binding domain, resulting in a stress protein that can tolerize dendritic cells in vitro. Introduced by gene gun, an expression plasmid encoding HSP70iQ435A can both prevent and treat autoimmune disease in mouse models of vitiligo. Here we tested the efficacy and safety of human HSP70iQ435A encoding DNA in human-like skin. We compared available methods of introducing DNA to porcine skin before subcloning HSP70iQ435A into clinically suitable vector pUMVC3. Next, vitiligo lesions in a substrain of Sinclair swine were treated with up to 5 mg plasmid DNA (N=6 lesions) or vehicle (N=4) weekly for 4 weeks to measure changes in lesional area, CD4 and CD8 T cell infiltration and serum HSP70 content and HSP70 antiserum titers in monthly biopsies and blood samples. After the gene gun, jet injection demonstrated greatest plasmid-encoded gene expression. Whereas untreated lesions continued to enlarge, significant repigmentation in response to HSP70iQ435A-encoding DNA lasted throughout the 6 months follow-up. Thus, serial samples of repigmenting, human-like skin and associated serum samples are now available to study the repigmentation process. Repigmentation was accompanied by an initial influx of CD4 T cells, followed by a significant reduction in perilesional T cells 3 months after treatment. Ta99+ melanocytes were 50% less plentiful but not absent from repigmenting skin, suggesting that melanocyte repopulation precedes skin melanization. No treatment-related changes in HSP70 or anti-HSP70 titers were observed. Importantly, treatment did not interfere with but rather supported melanoma immunosurveillance. These data encourage clinical testing of HSP70iQ435A.

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.

Activation of the DnaK-ClpB Complex is Regulated by the Properties of the Bound Substrate

The chaperone ClpB in bacteria is responsible for the reactivation of aggregated proteins in collaboration with the DnaK system. Association of these chaperones at the aggregate surface stimulates ATP hydrolysis, which mediates substrate remodeling. However, a question that remains unanswered is whether the bichaperone complex can be selectively activated by substrates that require remodeling. We find that large aggregates or bulky, native-like substrates activates the complex, whereas a smaller, permanently unfolded protein or extended, short peptides fail to stimulate it. Our data also indicate that ClpB interacts differently with DnaK in the presence of aggregates or small peptides, displaying a higher affinity for aggregate-bound DnaK, and that DnaK-ClpB collaboration requires the coupled ATPase-dependent remodeling activities of both chaperones. Complex stimulation is mediated by residues at the β subdomain of DnaK substrate binding domain, which become accessible to the disaggregase when the lid is allosterically detached from the β subdomain. Complex activation also requires an active NBD2 and the integrity of the M domain-ring of ClpB. Disruption of the M-domain ring allows the unproductive stimulation of the DnaK-ClpB complex in solution. The ability of the DnaK-ClpB complex to discrimínate different substrate proteins might allow its activation when client proteins require remodeling.

Glucose-regulated protein 78 substrate-binding domain alters its conformation upon EGCG inhibitor binding to nucleotide-binding domain: Molecular dynamics studies

Glucose-regulated protein 78 (GRP78), is overexpressed in glioblastoma, other tumors and during viral and bacterial infections, and so, it is postulated to be a key drug target. EGCG, an ATP-competitive natural inhibitor, inhibits GRP78 effect in glioblastoma. Structural basis of its action on GRP78 nucleotide-binding domain and selectivity has been investigated. We were interested in exploring the large-scale conformational movements travelling to substrate-binding domain via linker region. Conformational effects of EGCG inhibitor as well as ATP on full length GRP78 protein were studied using powerful MD simulations. Binding of EGCG decreases mobility of residues in SBDα lid region as compared to ATP-bound state and similar to apo state. The decreased mobility may prevent its opening and closing over SBDβ. This hindrance to SBDα subdomain movement, in turn, may reduce the binding of substrate peptide to SBDβ. EGCG binding folds the protein stably as opposed to ATP binding. Several results from EGCG binding simulations are similar to that of the apo state. Key insights from these results reveal that after EGCG binding upon competitive inhibition with ATP, GRP78 conformation may revert to that of inactive, apo state. Further, SBD may adopt a semi-open conformation unable to facilitate association of substrates.

The Misfolded Protein Client as a Determinant of Substrate Ubiquitination by the Hsp70/CHIP Complex

The E3 ubiquitin ligase CHIP (carboxy terminus of Hsc70 interacting protein) targets misfolded proteins chaperoned by heat shock protein 70 (Hsp70) for ubiquitination and subsequent proteasomal degradation. Hsp70 recruits and attempts to refold misfolded proteins, or “clients”, in iterative, ATP‐dependent cycles. In contrast, binding of Hsp70 to the tetratricopeptide repeat (TPR) domain of CHIP, allows for client proteins to be recruited for ubiquitination by CHIP. Ubiquitination of lysine residues on a client protein allows CHIP/Hsp70 complexes to promote the proteasomal degradation of the client. Thus, the CHIP/Hsp70 complex stands at the crossroads of degradative and refolding pathways of client proteins which is crucial for cellular protein quality control. The current knowledge in the field lacks a clear understanding of how the CHIP/Hsp70 complex executes its triage mechanism that directs misfolded proteins for either refolding or degradation. Recent models of ATP and ADP bound Hsp70 revealed the distances between the substrate binding domain (SBD) and the E2~ubiquitin conjugate active site as ~120 Å and ~60 Å respectively. This implies, with the dynamic conformational changes of Hsp70 complex, the ADP‐bound state of Hsp70 organizes the chaperoned ubiquitination complex by properly orienting the client with respect to CHIP which allows the client protein to span this 60 Å gap and accept the ubiquitin from the bound E2‐ubiquitin conjugate. Therefore, the distance between Hsp70 binding site and the active site of the E2~ubiquitin conjugate bound to CHIP plays a significant role as a determinant in the triage mechanism of CHIP/Hsp70. In this study, we focus on investigating the relationship between key distances within the CHIP/Hsp70/client complex and the ubiquitination activity of the CHIP/Hsp70 complex by utilizing in‐vitro ubiquitination assays of modified substrates.Support or Funding InformationNSF 15‐52113This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The ER Hsp70 Hspa13 Redirects an Amyloidogenic Protein to Aggregation

The conserved ER Hsp70 chaperone Hspa13/STCH is implicated in cancer and neurodegenerative diseases. While other Hsp70 proteins maintain proteostasis by promoting folding, degradation, or disaggregation of misfolded protein, the biological function of Hspa13 is unknown, in part because it lacks the substrate‐binding domain required for the function of other members of the Hsp70 class. We investigated the intracellular effect of Hspa13 overexpression on the kinetically stable, amyloidogenic protein transthyretin (TTR), finding that Hspa13 promotes the partitioning of TTR into high‐molecular weight, detergent‐resistant aggregates inside living cells. This function is ATP‐independent and opposed by the ER nucleotide exchange factor HYOU1, but unaffected by the primary ER Hsp70 BiP. These results suggest a role for Hspa13 in maintaining ER proteostasis by regulating the aggregation of amyloidogenic protein in the secretory pathway.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chloroplast SRP43 acts as a chaperone for glutamyl-tRNA reductase, the rate-limiting enzyme in tetrapyrrole biosynthesis

Assembly of light-harvesting complexes requires synchronization of chlorophyll (Chl) biosynthesis with biogenesis of light-harvesting Chl a/b-binding proteins (LHCPs). The chloroplast signal recognition particle (cpSRP) pathway is responsible for transport of nucleus-encoded LHCPs in the stroma of the plastid and their integration into the thylakoid membranes. Correct folding and assembly of LHCPs require the incorporation of Chls, whose biosynthesis must therefore be precisely coordinated with membrane insertion of LHCPs. How the spatiotemporal coordination between the cpSRP machinery and Chl biosynthesis is achieved is poorly understood. In this work, we demonstrate a direct interaction between cpSRP43, the chaperone that mediates LHCP targeting and insertion, and glutamyl-tRNA reductase (GluTR), a rate-limiting enzyme in tetrapyrrole biosynthesis. Concurrent deficiency for cpSRP43 and the GluTR-binding protein (GBP) additively reduces GluTR levels, indicating that cpSRP43 and GBP act nonredundantly to stabilize GluTR. The substrate-binding domain of cpSRP43 binds to the N-terminal region of GluTR, which harbors aggregation-prone motifs, and the chaperone activity of cpSRP43 efficiently prevents aggregation of these regions. Our work thus reveals a function of cpSRP43 in Chl biosynthesis and suggests a striking mechanism for posttranslational coordination of LHCP insertion with Chl biosynthesis.

Crystal structures of an archaeal chitinase ChiD and its ligand complexes.

Chitinase D (designated as Pc-ChiD) was found in a hyperthermophilic archaeon, Pyrococcus chitonophagus (previously described as Thermococcus chitonophagus), that was isolated from media containing only chitin as carbon source. Pc-ChiD displays chitinase activity and is thermostable at temperatures up to 95°C, suggesting its potential for industrial use. Pc-ChiD has a secretion signal peptide and two chitin-binding domains (ChBDs) in the N-terminal domain. However, the C-terminal domain shares no sequence similarity with previously identified saccharide-degrading enzymes and does not contain the DXDXE motif conserved in the glycoside hydrolase (GH) 18 family chitinases. To elucidate its overall structure and reaction mechanism, we determined the first crystal structures of Pc-ChiD, both in the ligand-free form and in complexes with substrates. Structure analyses revealed that the C-terminal domain of Pc-ChiD, Pc-ChiD(ΔBD), consists of a third putative substrate-binding domain, which cannot be predicted from the amino acid sequence, and a catalytic domain structurally similar to that found in not the GH18 family but the GH23 family. Based on the similarity with GH23 family chitinase, the catalytic residues of Pc-ChiD were predicted and confirmed by mutagenesis analyses. Moreover, the specific C-terminal 100 residues of Pc-ChiD are important to fix the putative substrate-binding domain next to the catalytic domain, contributing to the structure stability as well as the long chitin chain binding. Our findings reveal the structure of a unique archaeal chitinase that is distinct from previously known members of the GH23 family.

Substrate Binding Switches the Conformation at the Lynchpin Site in the Substrate-Binding Domain of Human Hsp70 to Enable Allosteric Interdomain Communication.

The stress-induced 70 kDa heat shock protein (Hsp70) functions as a molecular chaperone to maintain protein homeostasis. Hsp70 contains an N-terminal ATPase domain (NBD) and a C-terminal substrate-binding domain (SBD). The SBD is divided into the β subdomain containing the substrate-binding site (βSBD) and the α-helical subdomain (αLid) that covers the βSBD. In this report, the solution structures of two different forms of the SBD from human Hsp70 were solved. One structure shows the αLid bound to the substrate-binding site intramolecularly, whereas this intramolecular binding mode is absent in the other structure solved. Structural comparison of the two SBDs from Hsp70 revealed that client-peptide binding rearranges residues at the interdomain contact site, which impairs interdomain contact between the SBD and the NBD. Peptide binding also disrupted the inter-subdomain interaction connecting the αLid to the βSBD, which allows the binding of the αLid to the NBD. The results provide a mechanism for interdomain communication upon substrate binding from the SBD to the NBD via the lynchpin site in the βSBD of human Hsp70. In comparison to the bacterial ortholog, DnaK, some remarkable differences in the allosteric signal propagation among residues within the Hsp70 SBD exist.

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.

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.