Biochemical studies highlight determinants for metal selectivity in the Escherichia coli periplasmic solute binding protein NikA.

Nickel is an essential micronutrient for the survival of many microbes. On account of the toxicity of nickel and its scarcity in the environment, microbes have evolved specific systems for uptaking and delivering nickel to enzymes. NikA, the solute binding protein for the ATP-binding cassette (ABC) importer NikABCDE, plays a vital role in the nickel homeostasis of Escherichia coli by selectively binding nickel over other metals in the metabolically complex periplasm. While the endogenous ligand for NikA is known to be the Ni(II)-(L-His)2 complex, the molecular basis by which NikA selectively binds Ni(II)-(L-His)2 is unclear, especially considering that NikA can bind multiple metal-based ligands with comparable affinity. Here we show that, regardless of its promiscuous binding activity, NikA preferentially interacts with Ni(II)-(L-His)2, even over other metal-amino acid ligands with an identical coordination geometry for the metal. Replacing both the Ni(II) and the L-His residues in Ni(II)-(L-His)2 compromises binding of the ligand to NikA, in part because these alterations affect the degree by which NikA closes around the ligand. Replacing H416, the only NikA residue that ligates the Ni(II), with other potential metal-coordinating amino acids decreases the binding affinity of NikA for Ni(II)-(L-His)2 and compromises uptake of Ni(II) into E. coli cells, likely due to altered metal selectivity of the NikA mutants. Together, the biochemical and in vivo studies presented here define key aspects of how NikA selects for Ni(II)-(L-His)2 over other metal complexes, and can be used as a reference for studies into the metal selectivity of other microbial solute binding proteins.

Structure-based design of a photoswitchable affibody scaffold.

Photo-control of affinity reagents offers a general approach for high-resolution spatiotemporal control of diverse molecular processes. In an effort to develop general design principles for a photo-controlled affinity reagent, we took a structure-based approach to the design of a photoswitchable Z-domain, among the simplest of affinity reagent scaffolds. A chimera, designated Z-PYP, of photoactive yellow protein (PYP) and the Z-domain, was designed based on the concept of mutually exclusive folding. NMR analysis indicated that, in the dark, the PYP domain of the chimera was folded, and the Z-domain was unfolded. Blue light caused loss of structure in PYP and a two- to sixfold change in the apparent affinity of Z-PYP for its target as determined using size exclusion chromatography, UV-Vis based assays, and enyzme-linked immunosorbent assay (ELISA). A thermodynamic model indicated that mutations to decrease Z-domain folding energy would alter target affinity without loss of switching. This prediction was confirmed experimentally with a double alanine mutant in helix 3 of the Z-domain of the chimera (Z-PYP-AA) showing >30-fold lower dark-state binding and no loss in switching. The effect of decreased dark-state binding affinity was tested in a two-hybrid transcriptional control format and enabled pronounced light/dark differences in yeast growth in vivo. Finally, the design was transferable to the αZ-Taq affibody enabling tunable light-dependent binding both in vitro and in vivo to the Z-Taq target. This system thus provides a framework for the focused development of light switchable affibodies for a range of targets.

Structure of Ycf1p reveals the transmembrane domain TMD0 and the regulatory region of ABCC transporters.

ATP binding cassette (ABC) proteins typically function in active transport of solutes across membranes. The ABC core structure is composed of two transmembrane domains (TMD1 and TMD2) and two cytosolic nucleotide binding domains (NBD1 and NBD2). Some members of the C-subfamily of ABC (ABCC) proteins, including human multidrug resistance proteins (MRPs), also possess an N-terminal transmembrane domain (TMD0) that contains five transmembrane α-helices and is connected to the ABC core by the L0 linker. While TMD0 was resolved in SUR1, the atypical ABCC protein that is part of the hetero-octameric ATP-sensitive K+ channel, little is known about the structure of TMD0 in monomeric ABC transporters. Here, we present the structure of yeast cadmium factor 1 protein (Ycf1p), a homolog of human MRP1, determined by electron cryo-microscopy (cryo-EM). A comparison of Ycf1p, SUR1, and a structure of MRP1 that showed TMD0 at low resolution demonstrates that TMD0 can adopt different orientations relative to the ABC core, including a ∼145° rotation between Ycf1p and SUR1. The cryo-EM map also reveals that segments of the regulatory (R) region, which links NBD1 to TMD2 and was poorly resolved in earlier ABCC structures, interacts with the L0 linker, NBD1, and TMD2. These interactions, combined with fluorescence quenching experiments of isolated NBD1 with and without the R region, suggest how posttranslational modifications of the R region modulate ABC protein activity. Mapping known mutations from MRP2 and MRP6 onto the Ycf1p structure explains how mutations involving TMD0 and the R region of these proteins lead to disease.

HK97 gp74 Possesses an α-Helical Insertion in the ßßα Fold That Affects Its Metal Binding, cos Site Digestion, and In Vivo Activities.

The last gene in the genome of the bacteriophage HK97 encodes gp74, an HNH endonuclease. HNH motifs contain two conserved His residues and an invariant Asn residue, and they adopt a ßßα structure. gp74 is essential for phage head morphogenesis, likely because gp74 enhances the specific endonuclease activity of the HK97 terminase complex. Notably, the ability of gp74 to enhance the terminase-mediated cleavage of the phage cos site requires an intact HNH motif in gp74. Mutation of H82, the conserved metal-binding His residue in the HNH motif, to Ala abrogates gp74-mediated stimulation of terminase activity. Here, we present nuclear magnetic resonance (NMR) studies demonstrating that gp74 contains an α-helical insertion in the Ω-loop, which connects the two ß-strands of the ßßα fold, and a disordered C-terminal tail. NMR data indicate that the Ω-loop insert makes contacts to the ßßα fold and influences the ability of gp74 to bind divalent metal ions. Further, the Ω-loop insert and C-terminal tail contribute to gp74-mediated DNA digestion and to gp74 activity in phage morphogenesis. The data presented here enrich our molecular-level understanding of how HNH endonucleases enhance terminase-mediated digestion of the cos site and contribute to the phage replication cycle.IMPORTANCE This study demonstrates that residues outside the canonical ßßα fold, namely, the Ω-loop α-helical insert and a disordered C-terminal tail, regulate the activity of the HNH endonuclease gp74. The increased divalent metal ion binding when the Ω-loop insert is removed compared to reduced cos site digestion and phage formation indicates that the Ω-loop insert plays multiple regulatory roles. The data presented here provide insights into the molecular basis of the involvement of HNH proteins in phage DNA packing.

Intrinsically disordered regions regulate the activities of ATP binding cassette transporters.

ATP binding cassette (ABC) proteins are a large family of membrane proteins present in all kingdoms of life. These multi-domain proteins are comprised, at minimum, of two membrane-spanning domains (MSD1, MSD2) and two cytosolic nucleotide binding domains (NBD1, NBD2). ATP binding and hydrolysis at the NBDs enables ABC proteins to actively transport solutes across membranes, regulate activities of other proteins, or function as channels. Like most eukaryotic membrane proteins, ABC proteins contain intrinsically disordered regions (IDRs). These conformationally dynamic regions in ABC proteins possess residual structure, are sites of phosphorylation, and mediate protein-protein interactions. Here, we review the role of IDRs in regulating ABC protein activity.

Strategies for purification of the bacteriophage HK97 small and large terminase subunits that yield pure and homogeneous samples that are functional.

Packaging the viral genome in the head of double-stranded DNA viruses, such as bacteriophages, requires the activity of a terminase. The bacteriophage terminase consists of a small terminase subunit (TerS), which binds the viral DNA, and a large terminase subunit (TerL) that possesses the ATPase and nuclease activities for packaging the DNA in the phage head. Some phages require additional components for DNA packaging, such as the HNH endonuclease gp74 in the bacteriophage HK97. Gp74 enhances the activity of terminase-mediated digestion of the cohesive (cos) site that connects individual genomes in phage concatemeric DNA, a pre-requisite to DNA packaging, and this enhancement requires an intact HNH motif in gp74. Testing of whether gp74 alters the terminase DNA binding or enzymatic activities requires obtaining isolated samples of pure TerS and TerL, which has been challenging owing to the poor solubility of these proteins. To this end, we developed methods to obtain purified TerS and TerL proteins that are active. TerS is expressed solubly in E. coli as a fusion with SUMO, which can be removed during purification to yield a TerS nonamer (TerS9). Homogenous samples of a TerL monomer are also obtained, but the homogeneity of the sample depends on the solution conditions, as seen for other terminases. DNA binding, ATPase, and nuclease assays demonstrate that our preparations of TerS9 and TerL are functional, and that they also function with gp74. Purified TerS9 and TerL enable studies into the molecular basis by which gp74 regulates terminase activity in phage maturation.

Phosphorylation Alters the Residual Structure and Interactions of the Regulatory L1 Linker Connecting NBD1 to the Membrane-Bound Domain in SUR2B.

ATP-sensitive potassium (KATP) channels in vascular smooth muscle are comprised of four pore-forming Kir6.1 subunits and four copies of the sulfonylurea receptor 2B (SUR2B), which acts as a regulator of channel gating. Recent electron cryo-microscopy (cryo-EM) structures of the pancreatic KATP channel show a central Kir6.2 pore that is surrounded by the SUR1 subunits. Mutations in the L1 linker connecting the first membrane-spanning domain and the first nucleotide binding domain (NBD1) in SUR2B cause cardiac disease; however, this part of the protein is not resolved in the cryo-EM structures. Phosphorylation of the L1 linker, by protein kinase A, disrupts its interactions with NBD1, which increases the MgATP affinity of NBD1 and KATP channel gating. To elucidate the mode by which the L1 linker regulates KATP channels, we have probed the effects of phosphorylation on its structure and interactions using nuclear magnetic resonance (NMR) spectroscopy and other techniques. We demonstrate that the L1 linker is an intrinsically disordered region of SUR2B but possesses residual secondary and compact structure, both of which are disrupted with phosphorylation. NMR binding studies demonstrate that phosphorylation alters the mode by which the L1 linker interacts with NBD1. The data show that L1 linker residues with the greatest α-helical propensity also form the most stable interaction with NBD1, highlighting a hot spot within the L1 linker. This hot spot is the site of disease-causing mutations and is associated with other processes that regulate KATP channel gating. These data provide insights into the mode by which the phospho-regulatory L1 linker regulates KATP channels.

Molecular basis for the binding and modulation of V-ATPase by a bacterial effector protein.

Intracellular pathogenic bacteria evade the immune response by replicating within host cells. Legionella pneumophila, the causative agent of Legionnaires' Disease, makes use of numerous effector proteins to construct a niche supportive of its replication within phagocytic cells. The L. pneumophila effector SidK was identified in a screen for proteins that reduce the activity of the proton pumping vacuolar-type ATPases (V-ATPases) when expressed in the yeast Saccharomyces cerevisae. SidK is secreted by L. pneumophila in the early stages of infection and by binding to and inhibiting the V-ATPase, SidK reduces phagosomal acidification and promotes survival of the bacterium inside macrophages. We determined crystal structures of the N-terminal region of SidK at 2.3 Å resolution and used single particle electron cryomicroscopy (cryo-EM) to determine structures of V-ATPase:SidK complexes at ~6.8 Å resolution. SidK is a flexible and elongated protein composed of an α-helical region that interacts with subunit A of the V-ATPase and a second region of unknown function that is flexibly-tethered to the first. SidK binds V-ATPase strongly by interacting via two α-helical bundles at its N terminus with subunit A. In vitro activity assays show that SidK does not inhibit the V-ATPase completely, but reduces its activity by ~40%, consistent with the partial V-ATPase deficiency phenotype its expression causes in yeast. The cryo-EM analysis shows that SidK reduces the flexibility of the A-subunit that is in the 'open' conformation. Fluorescence experiments indicate that SidK binding decreases the affinity of V-ATPase for a fluorescent analogue of ATP. Together, these results reveal the structural basis for the fine-tuning of V-ATPase activity by SidK.

From ions to insulin.

Electron cryo-microscopy has revealed the three-dimensional structure of a potassium channel that has a central role in regulating the release of insulin from the pancreas.

Hyperinsulinism-Causing Mutations Cause Multiple Molecular Defects in SUR1 NBD1.

The sulfonylurea receptor 1 (SUR1) protein forms the regulatory subunit in ATP sensitive K+ (KATP) channels in the pancreas. SUR proteins are members of the ATP binding cassette (ABC) superfamily of proteins. Binding and hydrolysis of MgATP at the SUR nucleotide binding domains (NBDs) lead to channel opening. Pancreatic KATP channels play an important role in insulin secretion. SUR1 mutations that result in increased levels of channel opening ultimately inhibit insulin secretion and lead to neonatal diabetes. In contrast, SUR1 mutations that disrupt trafficking and/or decrease gating of KATP channels cause congenital hyperinsulinism, where oversecretion of insulin occurs even in the presence of low glucose levels. Here, we present data on the effects of specific congenital hyperinsulinism-causing mutations (G716V, R842G, and K890T) located in different regions of the first nucleotide binding domain (NBD1). Nuclear magnetic resonance (NMR) and fluorescence data indicate that the K890T mutation affects residues throughout NBD1, including residues that bind MgATP, NBD2, and coupling helices. The mutations also decrease the MgATP binding affinity of NBD1. Size exclusion and NMR data indicate that the G716V and R842G mutations cause aggregation of NBD1 in vitro, possibly because of destabilization of the domain. These data describe structural characterization of SUR1 NBD1 and shed light on the underlying molecular basis of mutations that cause congenital hyperinsulinism.

Phosphorylation-dependent changes in nucleotide binding, conformation, and dynamics of the first nucleotide binding domain (NBD1) of the sulfonylurea receptor 2B (SUR2B).

The sulfonylurea receptor 2B (SUR2B) forms the regulatory subunit of ATP-sensitive potassium (KATP) channels in vascular smooth muscle. Phosphorylation of the SUR2B nucleotide binding domains (NBD1 and NBD2) by protein kinase A results in increased channel open probability. Here, we investigate the effects of phosphorylation on the structure and nucleotide binding properties of NBD1. Phosphorylation sites in SUR2B NBD1 are located in an N-terminal tail that is disordered. Nuclear magnetic resonance (NMR) data indicate that phosphorylation of the N-terminal tail affects multiple residues in NBD1, including residues in the NBD2-binding site, and results in altered conformation and dynamics of NBD1. NMR spectra of NBD1 lacking the N-terminal tail, NBD1-ΔN, suggest that phosphorylation disrupts interactions of the N-terminal tail with the core of NBD1, a model supported by dynamic light scattering. Increased nucleotide binding of phosphorylated NBD1 and NBD1-ΔN, compared with non-phosphorylated NBD1, suggests that by disrupting the interaction of the NBD core with the N-terminal tail, phosphorylation also exposes the MgATP-binding site on NBD1. These data provide insights into the molecular basis by which phosphorylation of SUR2B NBD1 activates KATP channels.

Successful development and use of a thermodynamic stability screen for optimizing the yield of nucleotide binding domains.

ATP sensitive potassium (KATP) channels consist of four copies of a pore-forming inward rectifying potassium channel (Kir6.1 or Kir6.2) and four copies of a sulfonylurea receptor (SUR1, SUR2A, or SUR2B). SUR proteins are members of the ATP-binding cassette superfamily of proteins. Binding of ATP to the Kir6.x subunit mediates channel inhibition, whereas MgATP binding and hydrolysis at the SUR NBDs results in channel opening. Mutations in SUR1 and SUR2A NBDs cause diseases of insulin secretion and cardiac disorders, respectively, underlying the importance of studying the NBDs. Although purification of SUR2A NBD1 in a soluble form is possible, the lack of long-term sample stability of the protein in a concentrated form has precluded detailed studies of the protein aimed at gaining a molecular-level understanding of how SUR mutations cause disease. Here we use a convenient and cost-effective thermodynamic screening method to probe stabilizing conditions for SUR2A NBD1. Results from the screen are used to alter the purification protocol to allow for significantly increased yields of the purified protein. In addition, the screen provides strategies for long-term storage of NBD1 and generating NBD1 samples at high concentrations suitable for NMR studies. NMR spectra of NBD1 with MgAMP-PNP are of higher quality compared to using MgATP, indicating that MgAMP-PNP be used as the ligand in future NMR studies. The screen presented here can be expanded to using different additives and can be employed to enhance purification yields, sample life times, and storage of other low stability nucleotide binding domains, such as GTPases.

HNH proteins are a widespread component of phage DNA packaging machines.

The genome packaging reactions of tailed bacteriophages and herpes viruses require the activity of a terminase enzyme, which is comprised of large and small subunits. Phage genomes are replicated as linear concatemers composed of multiple copies of the genome joined end to end. As the terminase enzyme packages the genome into the phage capsid, it cleaves the DNA into single genome-length units. In this work, we show that the phage HK97 HNH protein, gp74, is required for the specific endonuclease activity of HK97 terminase and is essential for phage head morphogenesis. HNH proteins are a very common family of proteins generally associated with nuclease activity that are found in all kingdoms of life. We show that the activity of gp74 in terminase-mediated cleavage of the phage cos site relies on the presence of an HNH motif active-site residue, and that the large subunit of HK97 terminase physically interacts with gp74. Bioinformatic analysis reveals that the role of HNH proteins in terminase function is widespread among long-tailed phages and is uniquely required for the activity of the Terminase_1 family of large terminase proteins.

NMR and fluorescence studies of drug binding to the first nucleotide binding domain of SUR2A.

ATP sensitive potassium (K(ATP)) channels are composed of four copies of a pore-forming inward rectifying potassium channel (Kir6.1 or Kir6.2) and four copies of a sulfonylurea receptor (SUR1, SUR2A, or SUR2B) that surround the pore. SUR proteins are members of the ATP-binding cassette (ABC) superfamily of proteins. Binding of MgATP at the SUR nucleotide binding domains (NBDs) results in NBD dimerization, and hydrolysis of MgATP at the NBDs leads to channel opening. The SUR proteins also mediate interactions with K(ATP) channel openers (KCOs) that activate the channel, with KCO binding and/or activation involving residues in the transmembrane helices and cytoplasmic loops of the SUR proteins. Because the cytoplasmic loops make extensive interactions with the NBDs, we hypothesized that the NBDs may also be involved in KCO binding. Here, we report nuclear magnetic resonance (NMR) spectroscopy studies that demonstrate a specific interaction of the KCO pinacidil with the first nucleotide binding domain (NBD1) from SUR2A, the regulatory SUR protein in cardiac K(ATP) channels. Intrinsic tryptophan fluorescence titrations also demonstrate binding of pinacidil to SUR2A NBD1, and fluorescent nucleotide binding studies show that pinacidil binding increases the affinity of SUR2A NBD1 for ATP. In contrast, the KCO diazoxide does not interact with SUR2A NBD1 under the same conditions. NMR relaxation experiments and size exclusion chromatography indicate that SUR2A NBD1 is monomeric under the conditions used in drug binding studies. These studies identify additional binding sites for commonly used KCOs and provide a foundation for testing binding of drugs to the SUR NBDs.

The protein gp74 from the bacteriophage HK97 functions as a HNH endonuclease.

The last gene in the genome of the bacteriophage HK97 encodes the protein gp74. We present data in this article that demonstrates, for the first time, that gp74 possesses HNH endonuclease activity. HNH endonucleases are small DNA binding and digestion proteins characterized by two His residues and an Asn residue. We demonstrate that gp74 cleaves lambda phage DNA at multiple sites and that gp74 requires divalent metals for its endonuclease activity. We also present intrinsic tryptophan fluorescence data that show direct binding of Ni(2+) to gp74. The activity of gp74 in the presence of Ni(2+) is significantly decreased below neutral pH, suggesting the presence of one or more His residues in metal binding and/or DNA digestion. Surprisingly, this pH-dependence of activity is not seen with Zn(2+) , suggesting a different mode of binding of Zn(2+) and Ni(2+) . This difference in activity may result from binding of a second Zn(2+) ion by a putative zinc finger in gp74 in addition to binding of a Zn(2+) ion by the HNH motif. These studies define the biochemical function of gp74 as an HNH endonuclease and provide a platform for determining the role of gp74 in life cycle of the bacteriophage HK97.

The first nucleotide binding domain of the sulfonylurea receptor 2A contains regulatory elements and is folded and functions as an independent module.

The sulfonylurea receptor 2A (SUR2A) is an ATP-binding cassette (ABC) protein that forms the regulatory subunit of ATP-sensitive potassium (K(ATP)) channels in the heart. ATP binding and hydrolysis at the SUR2A nucleotide binding domains (NBDs) control gating of K(ATP) channels, and mutations in the NBDs that affect ATP hydrolysis and cellular trafficking cause cardiovascular disorders. To date, there is limited information on the SUR2A NBDs and the effects of disease-causing mutations on their structure and interactions. Structural and biophysical studies of NBDs, especially from eukaryotic ABC proteins like SUR2A, have been hindered by low solubility of the isolated domains. We hypothesized that the solubility of heterologously expressed SUR2A NBDs depends on the precise definition of the domain boundaries. Putative boundaries of SUR2A NBD1 were identified by structure-based sequence alignments and subsequently tested by exploring the solubility of SUR2A NBD1 constructs with different N and C termini. We have determined boundaries of SUR2A NBD1 that allow for soluble heterologous expression of the protein, producing a folded domain with ATP binding activity. Surprisingly, our alignment and screening data indicate that SUR2A NBD1 contains two putative, previously unidentified, regulatory elements: a large insert within the ß-sheet subdomain and a C-terminal extension. Our approach, which combines the use of structure-based sequence alignments and predictions of disordered regions combined with biochemical and biophysical studies, may be applied as a general method for developing suitable constructs of other NBDs of ABC proteins.

NMR spectroscopy to study the dynamics and interactions of CFTR.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a multi-domain membrane chloride channel whose activity is regulated by ATP at two nucleotide-binding domains (NBD1 and NBD2) and by phosphorylation of the regulatory (R) region. The NBDs and the R region have functionally relevant motions that are critical for channel gating. Nuclear magnetic resonance (NMR) spectroscopy is a highly useful technique for obtaining information on the structure and interactions of CFTR and is extremely powerful for probing dynamics. NMR approaches for studying CFTR are reviewed, using our previous NBD1 and the R region results to provide examples. These NMR data are yielding insights into the dynamic properties and interactions that facilitate normal CFTR regulation as well as pathological effects of mutations, including the most common disease mutant, deletion of F508 in NBD1.

The solution structure of the C-terminal Ig-like domain of the bacteriophage λ tail tube protein.

Immunoglobulin (Ig)-like domains are found frequently on the surface of tailed double-stranded DNA bacteriophages, yet their functional role remains obscure. Here, we have investigated the structure and function of the C-terminal Ig-like domain of gpV (gpV(C)), the tail tube protein of phage λ. This domain has been predicted through sequence similarity to be a member of the bacterial Ig-like domain 2 (Big_2) family, which is composed of more than 1300 phage and bacterial sequences. Using trypsin proteolysis, we have delineated the boundaries of gpV(C) and have shown that its removal reduces the biological activity of gpV by 100-fold; thus providing a definitive demonstration of a functional role for this domain. Determination of the solution structure of gpV(C) by NMR spectroscopy showed that it adopts a canonical Ig-like fold of the I-set class. This represents the first structure of a phage-encoded Ig-like domain and only the second structure of a Big_2 domain. Structural and sequence comparisons indicate that the gpV(C) structure is more representative of both the phage-encoded Big_2 domains and Big_2 domains in general than the other available Big_2 structure. Bioinformatics analyses have identified two conserved clusters of residues on the surface of gpV(C) that may be important in mediating the function of this domain.

NMR evidence for differential phosphorylation-dependent interactions in WT and DeltaF508 CFTR.

The most common cystic fibrosis (CF)-causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of Phe508 (DeltaF508) in the first of two nucleotide-binding domains (NBDs). Nucleotide binding and hydrolysis at the NBDs and phosphorylation of the regulatory (R) region are required for gating of CFTR chloride channel activity. We report NMR studies of wild-type and DeltaF508 murine CFTR NBD1 with the C-terminal regulatory extension (RE), which contains residues of the R region. Interactions of the wild-type NBD1 core with the phosphoregulatory regions, the regulatory insertion (RI) and RE, are disrupted upon phosphorylation, exposing a potential binding site for the first coupling helix of the N-terminal intracellular domain (ICD). Phosphorylation of DeltaF508 NBD1 does not as effectively disrupt interactions with the phosphoregulatory regions, which, along with other structural differences, leads to decreased binding of the first coupling helix. These results provide a structural basis by which phosphorylation of CFTR may affect the channel gating of full-length CFTR and expand our understanding of the molecular basis of the DeltaF508 defect.