Topological Analysis of Transthyretin Disassembly Mechanism: Surface-Induced Dissociation Reveals Hidden Reaction Pathways.

The proposed mechanism of fibril formation of transthyretin (TTR) involves self-assembly of partially unfolded monomers. However, the mechanism(s) of disassembly to monomer and potential intermediates involved in this process are not fully understood. In this study, native mass spectrometry and surface-induced dissociation (SID) are used to investigate the TTR disassembly mechanism(s) and the effects of temperature and ionic strength on the kinetics of TTR complex formation. Results from the SID of hybrid tetramers formed during subunit exchange provide strong evidence for a two-step mechanism whereby the tetramer dissociates to dimers that then dissociate to monomers. Also, the SID results uncovered a hidden pathway in which a specific topology of the hybrid tetramer is directly produced by assembly of dimers in the early steps of TTR disassembly. Implementation of SID to dissect protein topology during subunit exchange provides unique opportunities to gain unparalleled insight into disassembly pathways.

Membrane-anchored carbonic anhydrase IV interacts with monocarboxylate transporters via their chaperones CD147 and GP70.

Monocarboxylate transporters (MCTs) mediate the proton-coupled exchange of high-energy metabolites, including lactate and pyruvate, between cells and tissues. The transport activity of MCT1, MCT2, and MCT4 can be facilitated by the extracellular carbonic anhydrase IV (CAIV) via a noncatalytic mechanism. Combining physiological measurements in HEK-293 cells and Xenopus oocytes with pulldown experiments, we analyzed the direct interaction between CAIV and the two MCT chaperones basigin (CD147) and embigin (GP70). Our results show that facilitation of MCT transport activity requires direct binding of CAIV to the transporters chaperones. We found that this binding is mediated by the highly conserved His-88 residue in CAIV, which is also the central residue of the enzyme's intramolecular proton shuttle, and a charged amino acid residue in the Ig1 domain of the chaperone. Although the position of the CAIV-binding site in the chaperone was conserved, the amino acid residue itself varied among different species. In human CD147, binding of CAIV was mediated by the negatively charged Glu-73 and in rat CD147 by the positively charged Lys-73. In rat GP70, we identified the positively charged Arg-130 as the binding site. Further analysis of the CAIV-binding site revealed that the His-88 in CAIV can either act as H donor or H acceptor for the hydrogen bond, depending on the charge of the binding residue in the chaperone. Our results suggest that the CAIV-mediated increase in MCT transport activity requires direct binding between CAIV-His-88 and a charged amino acid in the extracellular domain of the transporter's chaperone.

Allostery revealed within lipid binding events to membrane proteins.

Membrane proteins interact with a myriad of lipid species in the biological membrane, leading to a bewildering number of possible protein-lipid assemblies. Despite this inherent complexity, the identification of specific protein-lipid interactions and the crucial role of lipids in the folding, structure, and function of membrane proteins is emerging from an increasing number of reports. Fundamental questions remain, however, regarding the ability of specific lipid binding events to membrane proteins to alter remote binding sites for lipids of a different type, a property referred to as allostery [Monod J, Wyman J, Changeux JP (1965) J Mol Biol 12:88-118]. Here, we use native mass spectrometry to determine the allosteric nature of heterogeneous lipid binding events to membrane proteins. We monitored individual lipid binding events to the ammonia channel (AmtB) from Escherichia coli, enabling determination of their equilibrium binding constants. We found that different lipid pairs display a range of allosteric modulation. In particular, the binding of phosphatidylethanolamine and cardiolipin-like molecules to AmtB exhibited the largest degree of allosteric modulation, inspiring us to determine the cocrystal structure of AmtB in this lipid environment. The 2.45-Å resolution structure reveals a cardiolipin-like molecule bound to each subunit of the trimeric complex. Mutation of a single residue in AmtB abolishes the positive allosteric modulation observed for binding phosphatidylethanolamine and cardiolipin-like molecules. Our results demonstrate that specific lipid-protein interactions can act as allosteric modulators for the binding of different lipid types to integral membrane proteins.

Structural and biophysical characterization of the α-carbonic anhydrase from the gammaproteobacterium Thiomicrospira crunogena XCL-2: insights into engineering thermostable enzymes for CO2 sequestration.

Biocatalytic CO2 sequestration to reduce greenhouse-gas emissions from industrial processes is an active area of research. Carbonic anhydrases (CAs) are attractive enzymes for this process. However, the most active CAs display limited thermal and pH stability, making them less than ideal. As a result, there is an ongoing effort to engineer and/or find a thermostable CA to fulfill these needs. Here, the kinetic and thermal characterization is presented of an α-CA recently discovered in the mesophilic hydrothermal vent-isolate extremophile Thiomicrospira crunogena XCL-2 (TcruCA), which has a significantly higher thermostability compared with human CA II (melting temperature of 71.9°C versus 59.5°C, respectively) but with a tenfold decrease in the catalytic efficiency. The X-ray crystallographic structure of the dimeric TcruCA shows that it has a highly conserved yet compact structure compared with other α-CAs. In addition, TcruCA contains an intramolecular disulfide bond that stabilizes the enzyme. These features are thought to contribute significantly to the thermostability and pH stability of the enzyme and may be exploited to engineer α-CAs for applications in industrial CO2 sequestration.

Structure and inhibition studies of a type II beta-carbonic anhydrase psCA3 from Pseudomonas aeruginosa.

Carbonic anhydrases (CAs) are metallo-enzymes that catalyze the reversible hydration of carbon dioxide into bicarbonate and a proton. The ß-class CAs (ß-CAs) are expressed in prokaryotes, fungi, plants, and more recently have been isolated in some animals. The ß-CA class is divided into two subclasses, termed type I and II, defined by pH catalytic activity profile and active site structural configuration. Type I ß-CAs display catalytic activity over a broad pH range (6.5-9.0) with the active site zinc tetrahedrally coordinated by three amino acids and a hydroxide/water. In contrast, type II ß-CAs are catalytically active only at a pH 8 and higher where they adopt a functional active site configuration like that of type I. However, below pH 8 they are conformationally self-inactivated by the addition of a fourth amino acid coordinating the zinc and thereby displacing the zinc bound solvent. We have determined the structure of psCA3, a type II ß-CA, isolated from Pseudomonas aeruginosa (P. aeruginosa) PAO1 at pH 8.3, in its open active state to a resolution of 1.9 Å. The active site zinc is coordinated by Cys42, His98, Cys101 and a water/hydroxide molecule. P. aeruginosa is a multi-drug resistant bacterium and displays intrinsic resistance to most of the currently used antibiotics; therefore, there is a need for new antibacterial targets. Kinetic data confirm that psCA3 belongs to the type II subclass and that sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid are micromolar inhibitors. In vivo studies identified that among six tested inhibitors representing sulfonamides, inorganic anions, and small molecules, acetazolamide has the most significant dose-dependent inhibitory effect on P. aeruginosa growth.

Structural and catalytic effects of proline substitution and surface loop deletion in the extended active site of human carbonic anhydrase II.

UNLABELLED: Bioengineering of a thermophilic enzyme starting from a mesophilic scaffold has proven to be a significant challenge, as several stabilizing elements have been proposed to be the foundation of thermal stability, including disulfide bridges, surface loop reduction, ionic pair networks, proline substitutions and aromatic clusters. This study emphasizes the effect of increasing the rigidity of human carbonic anhydrase II (HCA II; EC 4.2.1.1) via incorporation of proline residues at positions 170 and 234, which are located in surface loops that are able to accommodate restrictive main-chain conformations without rearrangement of the surrounding peptide backbone. Additionally, the effect of the compactness of HCA II was examined by deletion of a surface loop (residues 230-240) that had been previously identified as a possible source of thermal stability for the hyperthermophilic carbonic anhydrase isolated from the bacterium Sulfurihydrogenibium yellowstonense YO3AOP1. Differential scanning calorimetry analysis of these HCA II variants revealed that these structural modifications had a minimum effect on the thermal stability of the enzyme, while kinetic studies showed unexpected effects on the catalytic efficiency and proton transfer rates. X-ray crystallographic analysis of these HCA II variants showed that the electrostatic potential and configuration of the highly acidic loop (residues 230-240) play an important role in its high catalytic activity. Based on these observations and previous studies, a picture is emerging of the various components within the general structural architecture of HCA II that are key to stability. These elements may provide blueprints for rational thermal stability engineering of other enzymes. DATABASE: Structural data have been submitted to the Protein Data Bank under accession numbers 4QK1 (K170P), 4QK2 (E234P) and 4QK3 (Δ230-240).

Analysis of the binding moiety mediating the interaction between monocarboxylate transporters and carbonic anhydrase II.

Proton-coupled monocarboxylate transporters (MCTs) mediate the exchange of high energy metabolites like lactate between different cells and tissues. We have reported previously that carbonic anhydrase II augments transport activity of MCT1 and MCT4 by a noncatalytic mechanism, while leaving transport activity of MCT2 unaltered. In the present study, we combined electrophysiological measurements in Xenopus oocytes and pulldown experiments to analyze the direct interaction between carbonic anhydrase II (CAII) and MCT1, MCT2, and MCT4, respectively. Transport activity of MCT2-WT, which lacks a putative CAII-binding site, is not augmented by CAII. However, introduction of a CAII-binding site into the C terminus of MCT2 resulted in CAII-mediated facilitation of MCT2 transport activity. Interestingly, introduction of three glutamic acid residues alone was not sufficient to establish a direct interaction between MCT2 and CAII, but the cluster had to be arranged in a fashion that allowed access to the binding moiety in CAII. We further demonstrate that functional interaction between MCT4 and CAII requires direct binding of the enzyme to the acidic cluster (431)EEE in the C terminus of MCT4 in a similar fashion as previously shown for binding of CAII to the cluster (489)EEE in the C terminus of MCT1. In CAII, binding to MCT1 and MCT4 is mediated by a histidine residue at position 64. Taken together, our results suggest that facilitation of MCT transport activity by CAII requires direct binding between histidine 64 in CAII and a cluster of glutamic acid residues in the C terminus of the transporter that has to be positioned in surroundings that allow access to CAII.

Amiloride inhibits the initiation of Coxsackievirus and poliovirus RNA replication by inhibiting VPg uridylylation.

The mechanism of amiloride inhibition of Coxsackievirus B3 (CVB3) and poliovirus type 1 (PV1) RNA replication was investigated using membrane-associated RNA replication complexes. Amiloride was shown to inhibit viral RNA replication and VPgpUpU synthesis. However, the drug had no effect on polymerase elongation activity during either (-) strand or (+) strand synthesis. These findings indicated that amiloride inhibited the initiation of RNA synthesis by inhibiting VPg uridylylation. In addition, in silico binding studies showed that amiloride docks in the VPg binding site on the back of the viral RNA polymerase, 3D(pol). Since VPg binding at this site on PV1 3D(pol) was previously shown to be required for VPg uridylylation, our results suggest that amiloride inhibits VPg binding to 3D(pol). In summary, our findings are consistent with a model in which amiloride inhibits VPgpUpU synthesis and viral RNA replication by competing with VPg for binding to 3D(pol).

Structural elucidation of the hormonal inhibition mechanism of the bile acid cholate on human carbonic anhydrase II.

The carbonic anhydrases (CAs) are a family of mostly zinc metalloenzymes that catalyze the reversible hydration/dehydration of CO2 into bicarbonate and a proton. Human isoform CA II (HCA II) is abundant in the surface epithelial cells of the gastric mucosa, where it serves an important role in cytoprotection through bicarbonate secretion. Physiological inhibition of HCA II via the bile acids contributes to mucosal injury in ulcerogenic conditions. This study details the weak biophysical interactions associated with the binding of a primary bile acid, cholate, to HCA II. The X-ray crystallographic structure determined to 1.54 Šresolution revealed that cholate does not make any direct hydrogen-bond interactions with HCA II, but instead reconfigures the well ordered water network within the active site to promote indirect binding to the enzyme. Structural knowledge of the binding interactions of this nonsulfur-containing inhibitor with HCA II could provide the template design for high-affinity, isoform-specific therapeutic agents for a variety of diseases/pathological states, including cancer, glaucoma, epilepsy and osteoporosis.

The role of select subtype polymorphisms on HIV-1 protease conformational sampling and dynamics.

HIV-1 protease is an essential enzyme for viral particle maturation and is a target in the fight against HIV-1 infection worldwide. Several natural polymorphisms are also associated with drug resistance. Here, we utilized both pulsed electron double resonance, also called double electron-electron resonance, and NMR (15)N relaxation measurements to characterize equilibrium conformational sampling and backbone dynamics of an HIV-1 protease construct containing four specific natural polymorphisms commonly found in subtypes A, F, and CRF_01 A/E. Results show enhanced backbone dynamics, particularly in the flap region, and the persistence of a novel conformational ensemble that we hypothesize is an alternative flap orientation of a curled open state or an asymmetric configuration when interacting with inhibitors.

Preliminary X-ray crystallographic analysis of glutathione transferase zeta 1 (GSTZ1a-1a).

Glutathione transferase zeta 1 (GSTZ1-1) is a homodimeric enzyme found in the cytosol and mitochondrial matrix of the liver and other tissues. It catalyzes the glutathione-dependent isomerization of maleylacetoacetate to fumarylacetoacetate in the tyrosine catabolic pathway and can metabolize small halogenated carboxylic acids. GSTZ1a-1a crystals diffracted to a resolution of 3.1 Å and belonged to space group P1, with unit-cell parameters a = 42.0, b = 49.6, c = 54.6 Å, α = 82.9, ß = 69.9, γ = 73.4°, with a calculated Matthews coefficient of 2.1 Å(3) Da(-1) assuming a dimer in the crystallographic asymmetric unit. Refinement of the structure is currently in progress.

Catalytic mechanism of α-class carbonic anhydrases: CO2 hydration and proton transfer.

The carbonic anhydrases (CAs; EC 4.2.1.1) are a family of metalloenzymes that catalyze the reversible hydration of carbon dioxide (CO2) and dehydration of bicarbonate (HCO3 (-)) in a two-step ping-pong mechanism: [Formula: see text] CAs are ubiquitous enzymes and are categorized into five distinct classes (α, ß, γ, δ and ζ). The α-class is found primarily in vertebrates (and the only class of CA in mammals), ß is observed in higher plants and some prokaryotes, γ is present only in archaebacteria whereas the δ and ζ classes have only been observed in diatoms.The focus of this chapter is on α-CAs as the structure-function relationship is best understood for this class, in particular for humans. The reader is referred to other reviews for an overview of the structure and catalytic mechanism of the other CA classes. The overall catalytic site structure and geometry of α-CAs are described in the first section of this chapter followed by the kinetic studies, binding of CO2, and the proton shuttle network.

Structural study of interaction between brinzolamide and dorzolamide inhibition of human carbonic anhydrases.

Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes that catalyze the reversible hydration of carbon dioxide and bicarbonate. Their pivotal role in metabolism, ubiquitous nature, and multiple isoforms (CA I-XIV) has made CAs an attractive drug target in clinical applications. The usefulness of CA inhibitors (CAIs) in the treatment of glaucoma and epilepsy are well documented. In addition several isoforms of CAs (namely, CA IX) also serve as biological markers for certain tumors, and therefore they have the potential for useful applications in the treatment of cancer. This is a structural study on the binding interactions of the widely used CA inhibitory drugs brinzolamide (marketed as Azopt®) and dorzolamide (marketed as Trusopt®) with CA II and a CA IX-mimic, which was created via site-directed mutagenesis of CA II cDNA such that the active site resembles that of CA IX. Also the inhibition of CA II and CA IX and molecular docking reveal brinzolamide to be a more potent inhibitor among the other catalytically active CA isoforms compared to dorzolamide. The structures show that the tail end of the sulfonamide inhibitor is critical in forming stabilizing interactions that influence tight binding; therefore, for future drug design it is the tail moiety that will ultimately determine isoform specificity.

Structural, catalytic and stabilizing consequences of aromatic cluster variants in human carbonic anhydrase II.

The presence of aromatic clusters has been found to be an integral feature of many proteins isolated from thermophilic microorganisms. Residues found in aromatic cluster interact via π-π or C-H⋯π bonds between the phenyl rings, which are among the weakest interactions involved in protein stability. The lone aromatic cluster in human carbonic anhydrase II (HCA II) is centered on F226 with the surrounding aromatics F66, F95 and W97 located 12 Å posterior the active site; a location which could facilitate proper protein folding and active site construction. The role of F226 in the structure, catalytic activity and thermostability of HCA II was investigated via site-directed mutagenesis of three variants (F226I/L/W) into this position. The measured catalytic rates of the F226 variants via (18)O-mass spectrometry were identical to the native enzyme, but differential scanning calorimetry studies revealed a 3-4 K decrease in their denaturing temperature. X-ray crystallographic analysis suggests that the structural basis of this destabilization is via disruption and/or removal of weak C-H⋯π interactions between F226 to F66, F95 and W97. This study emphasizes the importance of the delicate arrangement of these weak interactions among aromatic clusters in overall protein stability.

Structural and catalytic characterization of a thermally stable and acid-stable variant of human carbonic anhydrase II containing an engineered disulfide bond.

The carbonic anhydrases (CAs) are a family of mostly zinc metalloenzymes that catalyze the reversible hydration of CO2 to bicarbonate and a proton. Recently, there has been industrial interest in utilizing CAs as biocatalysts for carbon sequestration and biofuel production. The conditions used in these processes, however, result in high temperatures and acidic pH. This unfavorable environment results in rapid destabilization and loss of catalytic activity in CAs, ultimately resulting in cost-inefficient high-maintenance operation of the system. In order to negate these detrimental industrial conditions, cysteines at residues 23 (Ala23Cys) and 203 (Leu203Cys) were engineered into a wild-type variant of human CA II (HCAII) containing the mutation Cys206Ser. The X-ray crystallographic structure of the disulfide-containing HCAII (dsHCAII) was solved to 1.77 Šresolution and revealed that successful oxidation of the cysteine bond was achieved while also retaining desirable active-site geometry. Kinetic studies utilizing the measurement of (18)O-labeled CO2 by mass spectrometry revealed that dsHCAII retained high catalytic efficiency, and differential scanning calorimetry showed acid stability and thermal stability that was enhanced by up to 14 K compared with native HCAII. Together, these studies have shown that dsHCAII has properties that could be used in an industrial setting to help to lower costs and improve the overall reaction efficiency.

Effects of cryoprotectants on the structure and thermostability of the human carbonic anhydrase II-acetazolamide complex.

Protein X-ray crystallography has seen a progressive shift from data collection at cool/room temperature (277-298 K) to data collection at cryotemperature (100 K) because of its ease of crystal preparation and the lessening of the detrimental effects of radiation-induced crystal damage, with 20-25%(v/v) glycerol (GOL) being the preferred choice of cryoprotectant. Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Comparative studies of crystal structure, kinetics, inhibition and thermostability were performed on CA II and its complex with AZM in the presence of either GOL or sucrose. These results suggest that even though the cryoprotectant GOL was previously shown to be directly bound in the active site and to interact with AZM, it affects neither the thermostability of CA II nor the binding of AZM in the crystal structure or in solution. However, addition of GOL does affect the kinetics of CA II, presumably as it displaces the water proton-transfer network in the active site.

Structural annotation of human carbonic anhydrases.

Carbonic anhydrases (CAs, EC 4.2.1.1) are a family of metalloenzymes that catalyze the reversible interconversion of CO(2) and HCO(3)(-). Of the 15 isoforms of human (h) α-CA, 12 are catalytic (hCAs I-IV, VA, VB, VI, VII, IX, XII-XIV). The remaining three acatalytic isoforms (hCAs VIII, X and XI) lack the active site Zn(2+) and are referred to as CA-related proteins (CA-RPs); however, their function remains elusive. Overall these isoforms are very similar to each other in structure but they differ in their expression and distribution. The favourable properties of hCA II such as fast kinetics, easy expression and purification, high solubility and intermediate heat resistance have made it an attractive candidate for numerous industrial applications. This review highlights the structural similarity and stability comparison among hCAs.

Carbonic anhydrases and their biotechnological applications.

The carbonic anhydrases (CAs) are mostly zinc-containing metalloenzymes which catalyze the reversible hydration/dehydration of carbon dioxide/bicarbonate. The CAs have been extensively studied because of their broad physiological importance in all kingdoms of life and clinical relevance as drug targets. In particular, human CA isoform II (HCA II) has a catalytic efficiency of 108 M-1 s-1, approaching the diffusion limit. The high catalytic rate, relatively simple procedure of expression and purification, relative stability and extensive biophysical studies of HCA II has made it an exciting candidate to be incorporated into various biomedical applications such as artificial lungs, biosensors and CO2 sequestration systems, among others. This review highlights the current state of these applications, lists their advantages and limitations, and discusses their future development.

Kinetic and structural characterization of thermostabilized mutants of human carbonic anhydrase II.

Carbonic anhydrases (CAs) are ubiquitous enzymes that catalyze the reversible hydration/dehydration of carbon dioxide/bicarbonate. As such, there is enormous industrial interest in using CA as a bio-catalyst for carbon sequestration and biofuel production. However, to ensure cost-effective use of the enzyme under harsh industrial conditions, studies were initiated to produce variants with enhanced thermostability while retaining high solubility and catalytic activity. Kinetic and structural studies were conducted to determine the structural and functional effects of these mutations. X-ray crystallography revealed that a gain in surface hydrogen bonding contributes to stability while retaining proper active site geometry and electrostatics to sustain catalytic efficiency. The kinetic profiles determined under a variety of conditions show that the surface mutations did not negatively impact the carbon dioxide hydration or proton transfer activity of the enzyme. Together these results show that it is possible to enhance the thermal stability of human carbonic anhydrase II by specific replacements of surface hydrophobic residues of the enzyme. In addition, combining these stabilizing mutations with strategic active site changes have resulted in thermostable mutants with desirable kinetic properties.

Global structure of HIV-1 neutralizing antibody IgG1 b12 is asymmetric.

Human antibody IgG1 b12 is one of the four antibodies known to neutralize a broad range of human immunodeficiency virus-1. The crystal structure of this antibody displayed an asymmetric disposition of the Fab arms relative to its Fc portion. Comparison of structures solved for other IgG1 antibodies led to a notion that crystal packing forces entrapped a "snap-shot" of different conformations accessible to this antibody. To elucidate global structure of this unique antibody, we acquired small-angle X-ray scattering data from its dilute solution. Data analysis indicated that b12 adopts a bilobal globular structure in solution with a radius of gyration and a maximum linear dimension of approximately 54 and approximately 180A, respectively. Extreme similarity between its solution and crystal structure concludes that non-flexible, asymmetric shape is an inherent property of this rare antibody.