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Human equilibrative nucleoside transporters represent a major pharmaceutical target for cardiac, cancer and viral therapies. Understanding the molecular basis for transport is crucial for the development of improved therapeutics through structure-based drug design. ENTs have been proposed to utilise an alternating access mechanism of action, similar to that of the major facilitator superfamily. However, ENTs lack functionally-essential features of that superfamily, suggesting that they may use a different transport mechanism. Understanding the molecular basis of their transport requires insight into diverse conformational states. Differences between intermediate states may be discrete and mediated by subtle gating interactions, such as salt bridges. We identified four variants of human equilibrative nucleoside transporter isoform 1 (hENT1) at the large intracellular loop (ICL6) and transmembrane helix 7 (TM7) that stabilise the apo-state (∆Tm 0.7–1.5°C). Furthermore, we showed that variants K263A (ICL6) and I282V (TM7) specifically stabilise the inhibitor-bound state of hENT1 (∆∆Tm 5.0 ± 1.7°C and 3.0 ± 1.8°C), supporting the role of ICL6 in hENT1 gating. Finally, we showed that, in comparison with wild type, variant T336A is destabilised by nitrobenzylthioinosine (∆∆Tm -4.7 ± 1.1°C) and binds it seven times worse. This residue may help determine inhibitor and substrate sensitivity. Residue K263 is not present in the solved structures, highlighting the need for further structural data that include the loop regions.

Inhibition of membrane‐bound pyrophosphatase (mPPase) with small molecules offer a new approach in the fight against pathogenic protozoan parasites. mPPases are absent in humans, but essential for many protists as they couple pyrophosphate hydrolysis to the active transport of protons or sodium ions across acidocalcisomal membranes. So far, only few nonphosphorus inhibitors have been reported. Here, we explore the chemical space around previous hits using a combination of screening and synthetic medicinal chemistry, identifying compounds with low micromolar inhibitory activities in the Thermotoga maritima mPPase test system. We furthermore provide early structure‐activity relationships around a new scaffold having a pyrazolo[1,5‐a]pyrimidine core. The most promising pyrazolo[1,5‐a]pyrimidine congener was further investigated and found to inhibit Plasmodium falciparum mPPase in membranes as well as the growth of P. falciparum in an ex vivo survival assay., Chemical space exploration: Novel low micromolar membrane‐bound pyrophosphatase (mPPase) inhibitors based on an exploratory screening against Thermotoga maritima mPPase followed by early SARs around a pyrazolo[1,5‐a]pyrimidine scaffold. Compound 17 a retained activity against Plasmodium falciparum mPPase in membranes, and inhibited parasite growth ex vivo.

The RET receptor tyrosine kinase plays a pivotal role in cell survival, proliferation, and differentiation, and its abnormal activation leads to cancers through receptor fusions or point mutations. Mutations that disrupt the disulfide network in the extracellular domain (ECD) of RET drive multiple endocrine neoplasia type 2A (MEN2A), a hereditary syndrome associated with the development of thyroid cancers. However, structural details of how specific mutations affect RET are unclear. Here, we present the first structural insights into the ECD of the RET(C634R) mutant, the most common mutation in MEN2A. Using electron microscopy, we demonstrate that the C634R mutation causes ligand-independent dimerization of the RET ECD, revealing an unusual tail-to-tail conformation that is distinct from the ligand-induced signaling dimer of WT RET. Additionally, we show that the RETC634R ECD dimer can form complexes with at least two of the canonical RET ligands and that these complexes form very different structures than WT RET ECD upon ligand binding. In conclusion, this structural analysis of cysteine-mutant RET ECD suggests a potential key mechanism of cancer induction in MEN2A, both in the absence and presence of its native ligands, and may offer new targets for therapeutic intervention.

Membrane-bound pyrophosphatases (mPPases) regulate energy homeostasis in pathogenic protozoan parasites and lack human homologues, which makes them promising targets in e.g. malaria. Yet only few nonphosphorus inhibitors have been reported so far. Here, we explore an isoxazole fragment hit, leading to the discovery of small mPPase inhibitors with 6-10 mu M IC50 values in the Thermotoga maritima test system. Promisingly, the compounds retained activity against Plasmodium falciparum mPPase in membranes and inhibited parasite growth.

Glutamate transporters play important roles in bacteria, archaea and eukaryotes. Their function in the mammalian central nervous system is essential for preventing excitotoxicity, and their dysregulation is implicated in many diseases, such as epilepsy and Alzheimer's. Elucidating their transport mechanism would further the understanding of these transporters and promote drug design as they provide compelling targets for understanding the pathophysiology of diseases and may have a direct role in the treatment of conditions involving glutamate excitotoxicity. This review outlines the insights into the transport cycle, uncoupled chloride conductance and modulation, as well as identifying areas that require further investigation.

In the accompanying paper, we described evolving a lipase to the point where variants were soluble, stable and capable of degrading C8 TAG and C8 esters. These variants were tested for their ability to survive in an environment that might be encountered in a washing machine. Unfortunately, they were inactivated both by treatment with a protease used in laundry detergents and by very low concentrations of sodium dodecyl sulfate (SDS). In addition, all the variants had very low levels of activity with triglycerides with long aliphatic chains and with naturally occurring oils, like olive oil. Directed evolution was used to select variants with enhanced properties. In the first 10 rounds of evolution, the primary screen was selected for variants capable of hydrolyzing olive oil whereas the secondary screen was selected for enhanced tolerance towards a protease and SDS. In the final six rounds of evolution, the primary and secondary screens identified variants that retained activity after treatment with SDS. Sixteen cycles of evolution gave variants with greatly enhanced lipolytic activity on substrates that had both long (C16 and C18) as well as short (C3 and C8) chains. We found variants that were stable for more than 3 hours in protease concentrations that rapidly degrade the wild-type enzyme. Enhanced tolerance towards SDS was found in variants that could break down naturally occurring lipid and resist protease attack. The amino acid changes that gave enhanced properties were concentrated in the cap domain responsible for substrate binding.

Characterizing the mycobacterial transporters involved in the uptake and/or catabolism of host-derived nutrients required by mycobacteria may identify novel drug targets against tuberculosis. Here, we identify and characterize a member of the amino acid-polyamine-organocation superfamily, a potential gamma-aminobutyric acid (GABA) transport protein, GabP, from Mycobacterium smegmatis. The protein was expressed to a level allowing its purification to homogeneity, and size exclusion chromatography coupled with multiangle laser light scattering (SEC-MALLS) analysis of the purified protein showed that it was dimeric. We showed that GabP transported gamma-aminobutyric acid both in vitro and when overexpressed in E. coli. Additionally, transport was greatly reduced in the presence of beta-alanine, suggesting it could be either a substrate or inhibitor of GabP. Using GabP reconstituted into proteoliposomes, we demonstrated that gamma-aminobutyric acid uptake is driven by the sodium gradient and is stimulated by membrane potential. Molecular docking showed that gamma-aminobutyric acid binds MsGabP, another Mycobacterium smegmatis putative GabP, and the Mycobacterium tuberculosis homologue in the same manner. This study represents the first expression, purification, and characterization of an active gamma-aminobutyric acid transport protein from mycobacteria. IMPORTANCE The spread of multidrug-resistant tuberculosis increases its global health impact in humans. As there is transmission both to and from animals, the spread of the disease also increases its effects in a broad range of animal species. Identifying new mycobacterial transporters will enhance our understanding of mycobacterial physiology and, furthermore, provides new drug targets. Our target protein is the gene product of msmeg_6196, annotated as GABA permease, from Mycobacterium smegmatis strain MC2 155. Our current study demonstrates it is a sodium-dependent GABA transporter that may also transport beta-alanine. As GABA may well be an essential nutrient for mycobacterial metabolism inside the host, this could be an attractive target for the development of new drugs against tuberculosis.

Membrane proteins have a range of crucial biological functions and are the target of about 60% of all prescribed drugs. For most studies, they need to be extracted out of the lipid-bilayer, e.g. by detergent solubilisation, leading to the loss of native lipids, which may disturb important protein-lipid/bilayer interactions and thus functional and structural integrity. Relipidation of membrane proteins has proven extremely successful for studying challenging targets, but the identification of suitable lipids can be expensive and laborious. Therefore, we developed a screen to aid the high-throughput identification of beneficial lipids. The screen covers a large lipid space and was designed to be suitable for a range of stability assessment methods. Here, we demonstrate its use as a tool for identifying stabilising lipids for three membrane proteins: a bacterial pyrophosphatase (Tm-PPase), a fungal purine transporter (UapA) and a human GPCR (A2AR). A2AR is stabilised by cholesteryl hemisuccinate, a lipid well known to stabilise GPCRs, validating the approach. Additionally, our screen also identified a range of new lipids which stabilised our test proteins, providing a starting point for further investigation and demonstrating its value as a novel tool for membrane protein research. The pre-dispensed screen will be made commercially available to the scientific community in future and has a number of potential applications in the field.

The receptor tyrosine kinase RET is essential in a variety of cellular processes. RET gain-of-function is strongly associated with several cancers, notably multiple endocrine neoplasia type 2A (MEN 2A), while RET loss-of-function causes Hirschsprung's disease and Parkinson's disease. To investigate the activation mechanism of RET as well as to enable drug development, over-expressed recombinant protein is needed for in vitro functional and structural studies. By comparing insect and mammalian cells expression of the RET extracellular domain (RETECD), we showed that the expression yields of RETECD using both systems were comparable, but mammalian cells produced monomeric functional RETECD, whereas RETECD expressed in insect cells was non-functional and multimeric. This was most likely due to incorrect disulfide formation. By fusing an Fc tag to the C-terminus of RETECD, we were able to produce, in HEK293T cells, dimeric oncogenic RETECD (C634R) for the first time. The protein remained dimeric even after cleavage of the tag via the cysteine disulfide, as in full-length RET in the context of MEN 2A and related pathologies. Our work thus provides valuable tools for functional and structural studies of the RET signaling system and its oncogenic activation mechanisms. (C) 2020 Published by Elsevier B.V.

A major obstacle to studying membrane proteins by biophysical techniques is the difficulty in producing sufficient amounts of materials for functional and structural studies. To overexpress the target membrane protein heterologously, especially an eukaryotic protein, a key step is to find the optimal host expression system and perform subsequent expression optimization. In this chapter, we describe protocols for screening membrane protein production using bacterial and insect cells, solubilization screening, large-scale production, and commonly used affinity chromatography purification methods. We discuss general optimization conditions, such as promoters and tags, and describe current techniques that can be used in any laboratory without specialized expensive equipment. Especially for insect cells, GFP fusions are particularly useful for localization and in-gel fluorescence detection of the proteins on SDS-PAGE. We give detailed protocols that can be used to screen the best expression and purification conditions for membrane protein study.

Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave pyrophosphate into two orthophosphate molecules, which is coupled with proton and/or sodium ion pumping across the membrane. Since no homologous proteins occur in animals and humans, mPPases are good candidates in the design of potential drug targets. Here we present a detailed protocol to screen for mPPase inhibitors utilizing the molybdenum blue reaction in a 96 well plate system. We use mPPase from the thermophilic bacterium Thermotoga maritima (TmPPase) as a model enzyme. This protocol is simple and inexpensive, producing a consistent and robust result. It takes only about one hour to complete the activity assay protocol from the start of the assay until the absorbance measurement. Since the blue color produced in this assay is stable for a long period of time, subsequent assay(s) can be performed immediately after the previous batch, and the absorbance can be measured later for all batches at once. The drawback of this protocol is that it is done manually and thus can be exhausting as well as require good skills of pipetting and time keeping. Furthermore, the arsenite-citrate solution used in this assay contains sodium arsenite, which is toxic and should be handled with necessary precautions.

Membrane integral pyrophosphatases (mPPases) are responsible for the hydrolysis of pyrophosphate. This enzymatic mechanism is coupled to the pumping of H+ or Na+ across membranes in a process that can be K+ dependent or independent. Understanding the movements and dynamics throughout the mPPase catalytic cycle is important, as this knowledge is essential for improving or impeding protein function. mPPases have been shown to play a crucial role in plant maturation and abiotic stress tolerance, and so have the potential to be engineered to improve plant survival, with implications for global food security. mPPases are also selectively toxic drug targets, which could be pharmacologically modulated to reduce the virulence of common human pathogens. The last few years have seen the publication of many new insights into the function and structure of mPPases. In particular, there is a new body of evidence that the catalytic cycle is more complex than originally proposed. There are structural and functional data supporting a mechanism involving half-of-the-sites reactivity, inter-subunit communication, and exit channel motions. A more advanced and in-depth understanding of mPPases has begun to be uncovered, leaving the field of research with multiple interesting avenues for further exploration and investigation.

Graphical abstract, Highlights • UspA1299–452 is a left-handed coiled-coil structure that follows TAA rules. • Structure of UspA1299–452 contains part of the long neck domain and of the stalk. • UspA1-C3d binding does not saturate at C3d physiological concentrations. • The binding constant as measured by thermophoresis is at least 140 μM. • Full-length proteins or other factors are important for UspA1-C3d interactions., The gram-negative bacterium Moraxella catarrhalis infects humans exclusively, causing various respiratory tract diseases, including acute otitis media in children, septicaemia or meningitis in adults, and pneumonia in the elderly. To do so, M. catarrhalis expresses virulence factors facilitating its entry and survival in the host. Among them are the ubiquitous surface proteins (Usps): A1, A2, and A2H, which all belong to the trimeric autotransporter adhesin family. They bind extracellular matrix molecules and inhibit the classical and alternative pathways of the complement cascade by recruiting complement regulators C3d and C4b binding protein. Here, we report the 2.5 Å resolution X-ray structure of UspA1299–452, which previous work had suggested contained the canonical C3d binding site found in both UspA1 and UspA2. We show that this fragment of the passenger domain contains part of the long neck domain (residues 299–336) and a fragment of the stalk (residues 337–452). The coiled-coil stalk is left-handed, with 7 polar residues from each chain facing the core and coordinating chloride ions or water molecules. Despite the previous reports of tight binding in serum-based assays, we were not able to demonstrate binding between C3d and UspA1299–452 using ELISA or biolayer interferometry, and the two proteins run separately on size-exclusion chromatography. Microscale thermophoresis suggested that the dissociation constant was 140.5 ± 8.4 μM. We therefore suggest that full-length proteins or other additional factors are important in UspA1-C3d interactions. Other molecules on the bacterial surface or present in serum may enhance binding of those two molecules.

Biochem. Soc. Trans. (2019) 47 (4) https://doi.org/10.1042/BST20190316 It has come to the attention of the authors that a citation for Figure 4 was omitted from the caption. The caption should read: Figure 4. Predicted anion permeation pathway. A Glt Ph monomer viewed from the trimerization interface. The residues R276 and M395 responsible for the anion selectivity are indicated. The orange circles represent ‘snapshots’ of the …

An enzyme must be soluble, stable, active and easy to produce to be useful in industrial applications. Not all enzymes possess these attributes. We set out to determine how many changes are required to convert an enzyme with poor properties into one that has useful properties. Lipase Lip3 from Drosophila melanogaster had been previously optimised for expression in Escherichia coli. The expression levels were good, but Lip3 was mainly insoluble with poor activity. Directed evolution was used to identify variants with enhanced activity along with improved solubility. Five variants and the wild-type (wt) enzyme were purified and characterised. The yield of the wt enzyme was just 2.2 mg/L of culture, while a variant, produced under the same conditions, gave 351 mg. The improvement of activity of the best variant was 200 times higher than that of the wt when the crude lysates were analysed using pNP-C8, but with purified protein, the improvement observed was 1.5 times higher. This means that most of the increase of activity is due to increase in solubility and stability. All the purified variants showed increased thermal stability compared with the wt enzyme that had a T1/2 of 37°C, while the mutant with P291L of 42.2°C and the mutant R7_47D with five mutations had a value of 52.9°C, corresponding to an improvement of 16°C. The improved variants had between five and nine changes compared with the wt enzyme. There were four changes that were found in all 30 final round variants for which sequences were obtained; three of these changes were found in the substrate-binding domain.

Membrane proteins represent one of the most important targets for pharmaceutical companies. Unfortunately, technical limitations have long been a major hindrance in our understanding of the function and structure of such proteins. Recent years have seen the refinement of classical approaches and the emergence of new technologies that have resulted in a significant step forward in the field of membrane protein research. This review summarizes some of the current techniques used for studying membrane proteins, with overall advantages and drawbacks for each method.

Membrane-bound pyrophosphatases (mPPases) are homodimeric integral membrane proteins that hydrolyse pyrophosphate into orthophosphates coupled to the active transport of protons or sodium ions across membranes. They occur in bacteria, archaea, plants, and protist parasites. As they are essential in protist parasites and there are no homologous proteins in animals and humans, these enzymes represent an excellent drug target for treating protistal diseases. Experimental screening to find drug candidates is an important step to discover new hit compounds. For that, a cheap, simple, and robust assay is needed. Here we report the application of the molybdenum blue reaction method for a medium throughput microplate activity assay of the hyperthermophilic bacterium Thermotoga maritima mPPase and the possible application of the assay to screen inhibitors of membrane-bound pyrophosphatases.

The main function of lysosomal proteins is to degrade cellular macromolecules. We purified a novel lysosomal protein to homogeneity from bovine kidneys. By gene annotation, this protein is defined as a bovine phospholipase B-like protein 1 (bPLBD1) and, to better understand its biological function, we solved its structure at 1.9 A resolution. We showed that bPLBD1 has uniform noncomplex-type N-glycosylation and that it localized to the lysosome. The first step in lysosomal protein transport, the initiation of mannose-6-phosphorylation by a N-acetylglucosamine-1-phosphotransferase, requires recognition of at least two distinct lysines on the protein surface. We identified candidate lysines by analyzing the structural and sequentially conserved N-glycosylation sites and lysines in bPLBD1 and in the homologous mouse PLBD2. Our model suggests that N408 is the primarily phosphorylated glycan, and K358 a key residue for N-acetylglucosamine-1-phosphotransferase recognition. Two other lysines, K334 and K342, provide the required second site for N-acetylglucosamine-1-phosphotransferase recognition. bPLBD1 is an N-terminal nucleophile (Ntn) hydrolase. By comparison with other Ntn-hydrolases, we conclude that the acyl moiety of PLBD1 substrate must be small to fit the putative binding pocket, whereas the space for the rest of the substrate is a large open cleft. Finally, as all the known substrates of Ntn-hydrolases have amide bonds, we suggest that bPLBD1 may be an amidase or peptidase instead of lipase, explaining the difficulty in finding a good substrate for any members of the PLBD family. Proteins 2014; 82:300–311. © 2013 Wiley Periodicals, Inc.

Nucleoside transporters (NTs) play critical biological roles in humans, and to understand the molecular mechanism of nucleoside transport requires high-resolution structural information. However, the main bottleneck for structural analysis of NTs is the production of pure, stable, and high quality native protein for crystallization trials. Here we report a novel membrane protein expression and purification strategy, including construction of a high-yield membrane protein expression vector, and a new and fast purification protocol for NTs. The advantages of this strategy are the improved time efficiency, leading to high quality, active, stable membrane proteins, and the efficient use of reagents and consumables. Our strategy might serve as a useful point of reference for investigating NTs and other membrane proteins by clarifying the technical points of vector construction and improvements of membrane protein expression and purification. © 2016 Hao, Thomsen, Postis, Lesiuk, Sharples, Wang, Bartlam and Goldman.

Despite the great importance of membrane proteins, structural and functional studies of these proteins present major challenges. A significant hurdle is the extraction of the functional protein from its natural lipid membrane. Traditionally achieved with detergents, purification procedures can be costly and time consuming. A critical flaw with detergent approaches is the removal of the protein from the native lipid environment required to maintain functionally stable protein. This protocol describes the preparation of styrene maleic acid (SMA) co-polymer to extract membrane proteins from prokaryotic and eukaryotic expression systems. Successful isolation of membrane proteins into SMA lipid particles (SMALPs) allows the proteins to remain with native lipid, surrounded by SMA. We detail procedures for obtaining 25 g of SMA (4 d); explain the preparation of protein-containing SMALPs using membranes isolated from Escherichia coli (2 d) and control protein-free SMALPS using E. coli polar lipid extract (1-2 h); investigate SMALP protein purity by SDS-PAGE analysis and estimate protein concentration (4 h); and detail biophysical methods such as circular dichroism (CD) spectroscopy and sedimentation velocity analytical ultracentrifugation (svAUC) to undertake initial structural studies to characterize SMALPs (∼2 d). Together, these methods provide a practical tool kit for those wanting to use SMALPs to study membrane proteins.

AMIGO-1 is the parent member of a novel family of three cell surface leucine-rich repeat (LRR) proteins. Its expression is induced by the binding of HMGB1 ( h igh- m obility g roup b ox 1 protein) to RAGE ( r eceptor for a dvanced g lycation e nd products) on neurons. Binding of HMGB1 to RAGE is known to have a direct effect on cellular growth regulation and mobility, and AMIGO-1 directly supports growth of neuronal processes and fasciculation of neurites. In addition, the second member of the AMIGO-family, AMIGO-2, has been implicated in adhesion of tumor cells in adenocarcinoma and survival of neurons. We have determined the crystal structure of AMIGO-1 at 2.0 A resolution, which reveals a typical cell surface LRR domain arrangement with N- and C-terminal capping domains with disulfide bridges, followed by a C2-type Ig domain. AMIGO-1 is a dimer, with the LRR regions forming the dimer interface, and sequence conservation analysis and static light-scattering measurements suggest that all three AMIGO family proteins form similar dimers. Based on the AMIGO-1 structure, we have also modeled AMIGO-2 and present small-angle X-ray scattering data on AMIGO-2 and AMIGO-3. Our mutagenesis studies show that AMIGO-1 dimerization is necessary for proper cell surface expression and thus probably for proper or stable folding in the endoplastic reticulum and for the function of the protein. Based on the data presented earlier, we also suggest that dimerization through the LRR–LRR interface is likely to be involved in cell–cell adhesion by AMIGO-1, while extensive glycosylation may have a role.

Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain. By reducing oxygen to water, it generates a proton gradient across the mitochondrial or bacterial membrane. Recently, two independent X-ray crystallographic studies ((Aoyama et al. Proc. Natl. Acad. Sci. USA 106 (2009) 2165–2169) and (Koepke et al. Biochim. Biophys. Acta 1787 (2009) 635–645)), suggested that a peroxide dianion might be bound to the active site of oxidized CcO. We have investigated this hypothesis by combining quantum chemical calculations with a re-refinement of the X-ray crystallographic data and optical spectroscopic measurements. Our data suggest that dianionic peroxide, superoxide, and dioxygen all form a similar superoxide species when inserted into a fully oxidized ferric/cupric binuclear site (BNC). We argue that stable peroxides are unlikely to be confined within the oxidized BNC since that would be expected to lead to bond splitting and formation of the catalytic P intermediate. Somewhat surprisingly, we find that binding of dioxygen to the oxidized binuclear site is weakly exergonic, and hence, the observed structure might have resulted from dioxygen itself or from superoxide generated from O2 by the X-ray beam. We show that the presence of O2 is consistent with the X-ray data. We also discuss how other structures, such as a mixture of the aqueous species (H2O + OH− and H2O) and chloride fit the experimental data.

The RadA intein from the hyperthermophilic archaebacterium Pyrococcus horikoshii was cloned, expressed and purified for subsequent structure determination. The protein crystallized rapidly in several conditions. The best crystals, which diffracted to 1.75 Å resolution, were harvested from drops consisting of 0.1 M HEPES pH 7.5, 3.0 M NaCl and were cryoprotected with Paratone-N before flash-cooling. The collected data were processed in the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 58.1, b = 67.4, c = 82.9 Å. Molecular replacement with Rosetta using energy- and density-guided structure optimization provided the initial solution, which is currently under refinement.

Nucleotide-binding cystathionine β-synthase (CBS) domains serve as regulatory units in numerous proteins distributed in all kingdoms of life. However, the underlying regulatory mechanisms remain to be established. Recently, we described a subfamily of CBS domain-containing pyrophosphatases (PPases) within family II PPases. Here, we express a novel CBS-PPase from Clostridium perfringens (CPE2055) and show that the enzyme is inhibited by AMP and activated by a novel effector, diadenosine 5′,5-P1,P4-tetraphosphate (AP4A). The structures of the AMP and AP4A complexes of the regulatory region of C. perfringens PPase (cpCBS), comprising a pair of CBS domains interlinked by a DRTGG domain, were determined at 2.3 A resolution using X-ray crystallography. The structures obtained are the first structures of a DRTGG domain as part of a larger protein structure. The AMP complex contains two AMP molecules per cpCBS dimer, each bound to a single monomer, whereas in the activator-bound complex, one AP4A molecule bridges two monomers. In the nucleotide-bound structures, activator binding induces significant opening of the CBS domain interface, compared with the inhibitor complex. These results provide structural insight into the mechanism of CBS-PPase regulation by nucleotides.

Factor H (FH) regulates the alternative pathway of complement in plasma and mediates discrimination of cellular surfaces to alternative pathway activators and non-activators. The carboxyl-terminal domains 19 and 20 of FH are essential in target discrimination and are known to contain binding sites for the C3d part of C3b, heparin, and endothelial cells. Mutations in FH19-20 are frequently found in patients with atypical haemolytic uremic syndrome (aHUS). Most aHUS-associated and some other mutations have been shown to lead to impaired binding to C3d and C3b by the recombinant FH19-20 fragment. Most of these mutated residues, such as R1203, are located close to each other in domain 20 but some, such as Q1139, are located in domain 19. We generated mutant proteins Q1139A and R1203A of FH19-20 and showed that their binding to C3d and C3b was clearly impaired. To show that the effects on C3d/C3b binding are due to direct interactions rather than structural changes, we solved the X-ray crystal structures of the R1203A and Q1139A mutant proteins at 1.65 and 2.0A, respectively. Neither of the mutations caused any overall structural changes in FH19-20. It is thus evident that Q1139 in domain 19 and R1203 in domain 20 are directly involved in binding to the C3d part of C3b and therefore both the domains are involved in the interaction with C3d and C3b. This explains why several aHUS-associated FH mutations are found within domain 19 in addition to domain 20.

Peroxiredoxin II was cloned from mouse B cells into pCold 1 expression vector and produced as a His-tagged recombinant protein in Escherichia coli. A ring form was isolated by gel filtration. A crystal obtained by the sitting-drop vapour-diffusion method diffracted to 1.77 A resolution at 100 K. The crystal belonged to space group P2(1)2(1)2, with unit-cell parameters a = 117.4, b = 133.9, c = 139.1 A. The asymmetric unit is expected to contain six dimers of peroxiredoxin II, with a corresponding solvent content of 39.3%. Peaks in the native Patterson function together with pseudo-systematic absences suggested that the crystals suffered from severe translational pseudosymmetry.

The Hsp-organizing protein (HOP) binds to the C termini of the chaperones Hsp70 and Hsp90, thus bringing them together so that substrate proteins can be passed from Hsp70 to Hsp90. Because Hsp90 is essential for the correct folding and maturation of many oncogenic proteins, it has become a significant target for anti-cancer drug design. HOP binds to Hsp70 and Hsp90 via two independent tetratricopeptide (TPR) domains, TPR1 and TPR2A, respectively. We have analyzed ligand binding using Poisson-Boltzmann continuum electrostatic calculations, free energy perturbation, molecular dynamics simulations, and site-directed mutagenesis to delineate the contribution of different interactions to the affinity and specificity of the TPR-peptide interactions. We found that continuum electrostatic calculations could be used to guide protein design by removing unfavorable interactions to increase binding affinity, with an 80-fold increase in affinity for TPR2A. Contributions at buried charged residues, however, were better predicted by free energy perturbation calculations. We suggest using a combination of the two approaches for increasing the accuracy of results, with free energy perturbation calculations used only at selected buried residues of the ligand binding pocket. Finally we present the crystal structure of TPR2A in complex with its non-cognate Hsp70 ligand, which provides insight on the origins of specificity in TPR domain-peptide recognition.

We have solved the structures of mammalian mesencephalic astrocyte-derived neurotrophic factor (MANF) and conserved dopamine neurotrophic factor (CDNF). CDNF protects and repairs midbrain dopaminergic neurons in vivo; MANF supports their survival in culture and is also cytoprotective against endoplasmic reticulum (ER) stress. Neither protein structure resembles any known growth factor but the N-terminal domain is a saposin-like lipid-binding domain. MANF and CDNF may thus bind lipids or membranes. Consistent with this, there are two patches of conserved lysines and arginines. The natively unfolded MANF C-terminus contains a CKGC disulphide bridge, such as reductases and disulphide isomerases, consistent with a role in ER stress response. The structure thus explains why MANF and CDNF are bifunctional; neurotrophic activity may reside in the N-terminal domain and ER stress response in the C-terminal domain. Finally, we identified three changes, (MANF)I10-->K(CDNF), (MANF)E79-->M(CDNF) and (MANF)K88-->L(CDNF), that may account for the biological differences between the proteins.

Glial cell line-derived neurotrophic factor (GDNF), a neuronal survival factor, binds its co-receptor GDNF family receptor α1 (GFRα1) in a 2:2 ratio and signals through the receptor tyrosine kinase RET. We have solved the GDNF2·GFRα12 complex structure at 2.35 A resolution in the presence of a heparin mimic, sucrose octasulfate. The structure of our GDNF2·GFRα12 complex and the previously published artemin2·GFRα32 complex are unlike in three ways. First, we have experimentally identified residues that differ in the ligand-GFRα interface between the two structures, in particular ones that buttress the key conserved ArgGFRα-Gluligand-ArgGFRα interaction. Second, the flexible GDNF ligand “finger” loops fit differently into the GFRαs, which are rigid. Third, and we believe most importantly, the quaternary structure of the two tetramers is dissimilar, because the angle between the two GDNF monomers is different. This suggests that the RET-RET interaction differs in different ligand2-co-receptor2-RET2 heterohexamer complexes. Consistent with this, we showed that GDNF2·GFRα12 and artemin2·GFRα32 signal differently in a mitogen-activated protein kinase assay. Furthermore, we have shown by mutagenesis and enzyme-linked immunosorbent assays of RET phosphorylation that RET probably interacts with GFRα1 residues Arg-190, Lys-194, Arg-197, Gln-198, Lys-202, Arg-257, Arg-259, Glu-323, and Asp-324 upon both domains 2 and 3. Interestingly, in our structure, sucrose octasulfate also binds to the Arg190-Lys202 region in GFRα1 domain 2. This may explain how GDNF·GFRα1 can mediate cell adhesion and how heparin might inhibit GDNF signaling through RET.

The Yersinia adhesin A (YadA) is a collagen-binding trimeric autotransporter of Yersinia enterocolitica, an enteropathogen that causes a range of gastroenteric and systemic diseases, and YadA is essential for Y. enterocolitica virulence. Although previous studies suggest a specific binding site in collagen for YadA, we found that recombinant YadA binds to both major cyanogen bromide fragments of collagen type II and the collagen-like model peptide (Pro-Hyp-Gly)(10) [(POG)(10)]. To further characterise the YadA-collagen interaction, we investigated the binding of YadA to (POG)(10) and three other model peptides, (Pro-Pro-Gly)(10) which lacks the hydroxyl groups of (POG)(10), T3-785 which contains a stretch of the collagen type III sequence and Gly(-) which is similar to (POG)(10) but lacks the central glycine. All the peptides except Gly(-) adopt a collagen-like triple-helical conformation at room temperature. All three triple-helical peptides bound to YadA, with (POG)(10) being the tightest, whereas binding of Gly(-) was hardly detectable. The affinity of (POG)(10) for YadA was 0.28 microM by isothermal titration calorimetry and 0.17 microM by surface plasmon resonance (SPR), similar to that of collagen type I. Our results show that a collagen-like triple-helical conformation, strengthened by the presence of hydroxyproline residues, is both necessary and sufficient for YadA binding.

All UDP-glucuronosyltransferase enzymes (UGTs) share a common cofactor, UDP-glucuronic acid (UDP-GlcUA). The binding site for UDP-GlcUA is localized to the C-terminal domain of UGTs on the basis of amino acid sequence homology analysis and crystal structures of glycosyltransferases, including the C-terminal domain of human UGT2B7. We hypothesized that the (393)DQMDNAK(399) region of human UGT1A10 interacts with the glucuronic acid moiety of UDP-GlcUA. Using site-directed mutagenesis and enzymatic analysis, we demonstrated that the D393A mutation abolished the glucuronidation activity of UGT1A10 toward all substrates. The effects of the alanine mutation at Q(394),D(396), and K(399) on glucuronidation activities were substrate-dependent. Previously, we examined the importance of these residues in UGT2B7. Although D(393) (D(398) in UGT2B7) is similarly critical for UDP-GlcUA binding in both enzymes, the effects of Q(394) (Q(399) in UGT2B7) to Ala mutation on activity were significant but different between UGT1A10 and UGT2B7. A model of the UDP-GlcUA binding site suggests that the contribution of other residues to cosubstrate binding may explain these differences between UGT1A10 and UGT2B7. We thus postulate that D(393) is critical for the binding of glucuronic acid and that proximal residues, e.g., Q(394) (Q(399) in UGT2B7), play a subtle role in cosubstrate binding in UGT1A10 and UGT2B7. Hence, this study provides important new information needed for the identification and understanding of the binding sites of UGTs, a major step forward in elucidating their molecular mechanism.

Collagen IX, located on the surface of collagen fibrils, is crucial for cartilage integrity and stability. The N-terminal NC4 domain of the alpha1(IX) chain is probably important in this because it interacts with various macromolecules such as proteoglycans and cartilage oligomeric matrix protein. At least 17 distinct collagen polypeptides carry an NC4-like unit near their N terminus, but this report, describing the crystal structure of NC4 at 1.8-A resolution, represents the first atomic level structure for these domains. The structure is similar to previously characterized laminin-neurexin-sex hormone binding globulin (LNS) structures, dominated by an antiparallel beta-sheet sandwich. In addition, a zinc ion was found in a position similar to that of the metal binding site of other LNS domains. A partial backbone NMR assignment of NC4 was obtained and utilized in NMR titration studies to investigate the zinc binding in solution state and to quantitate the affinity of metal binding. The K(d) of 11.5 mM suggests a regulatory rather than a structural role for zinc in solution. NMR titration with a heparin tetrasaccharide revealed the presence of a secondary binding site for heparin on NC4, showing structural and functional conservation with thrombospondin-1, but a markedly reduced affinity for the ligand. Also the overall arrangement of the N and C termini of NC4 resembles most closely the N-terminal domain of thrombospondin-1, distinguishing the two from the majority of the published LNS structures.

UDP-glucuronosyltransferases (UGTs) play important roles in the metabolism, detoxification,and clearance of many different xenobiotics, including drugs and endogenous compounds. Structural information about these membrane-bound enzymes of the endoplasmic reticulum is limited. We do not know the identity or the location of the key residues for catalysis and binding of the aglycone substrate and the cosubstrate UDP-glucuronic acid (UDPGA). One suggestion was that His371 (UGT1A6 numbering) is the "catalytic base" that deprotonates the phenol group. We have now re-examined this hypothesis by analyzing the activities of the corresponding mutants, 6H371A (in UGT1A6) and the 9H369A (in UGT1A9). The K(m) values of mutant 6H371A for scopoletin and UDPGA were higher by 4- and 11-fold, respectively, than in UGT1A6. The K(d) for the enzyme-UDPGA complex, derived from bisubstrate kinetics, was about 9-fold higher in 6H371A than in UGT1A6, indicating severely impaired cosubstrate binding by the mutant. The effect of mutation on V(max) was large in UGT1A6 but variable in UGT1A9, suggesting that His371 does not play the catalytic role previously hypothesized. In both UGTs, the E379A mutation (UGT1A6 numbering) had an effect similar to that of the H371A mutations. A homology model of the putative UDPGA binding region of UGT1A6 was built using distant homologous protein structures from the "GT1 class." The combined results of activity determinations, kinetic analyses, and modeling strongly suggest that His371 and Glu379 are directly involved in UDPGA binding but are not the general acid or general base.

We solved the crystal structure of Streptococcus agalactiae serine/threonine phosphatase (SaSTP) using a combination of single-wavelength anomalous dispersion phasing and molecular replacement. The overall structure resembles that of previously characterized members of the PPM/PP2C STP family. The asymmetric unit contains four monomers and we observed two novel conformations for the flap domain among them. In one of these conformations, the enzyme binds three metal ions, whereas in the other it binds only two. The three-metal ion structure also has the active site arginine in a novel conformation. The switch between the two- and three-metal ion structures appears to be binding of another monomer to the active site of STP, which promotes binding of the third metal ion. This interaction may mimic the binding of a product complex, especially since the motif binding to the active site contains a serine residue aligning remarkably well with the phosphate found in the human STP structure.

We have determined the structures of the wild type and seven active site variants of yeast inorganic pyrophosphatase (PPase) in the presence of Mg 2+ and phosphate, providing the first complete structural description of its catalytic cycle. PPases catalyze the hydrolysis of pyrophosphate and require four divalent metal cations for catalysis; magnesium provides the highest activity. The crystal form chosen contains two monomers in the asymmetric unit, corresponding to distinct catalytic intermediates. In the "closed" wild-type active site, one of the two product phosphates has already dissociated, while the D115E variant "open" conformation is of the hitherto unobserved two-phosphate and two-"bridging" water active site. The mutations affect metal binding and the hydrogen bonding network in the active site, allowing us to explain the effects of mutations. For instance, in Y93F, F93 binds in a cryptic hydrophobic pocket in the absence of substrate, preserving hydrogen bonding in the active site and leading to relatively small changes in solution properties. This is not true in the presence of substrate, when F93 is forced back into the active site. Such subtle changes underline how low the energy barriers are between thermodynamically favorable conformations of the enzyme. The structures also allow us to associate metal binding constants to specific sites. Finally, the wild type and the D152E variant contain a phosphate ion adjacent to the active site, showing for the first time how product is released through a channel of flexible cationic side chains.

3-carboxy-cis,cis-muconate lactonizing enzymes participate in the protocatechuate branch of the 3-oxoadipate pathway of various aerobic bacteria. The gene encoding a 3-carboxy-cis,cis-muconate lactonizing enzyme (pcaB1S2) was cloned from a gene cluster involved in protocatechuate degradation by Agrobacterium radiobacter strain S2. This gene encoded for a 3-carboxy-cis,cis-muconate lactonizing enzyme of 353 amino acids - significantly smaller than all previously studied 3-carboxy-cis,cis-muconate lactonizing enzymes. This enzyme, ArCMLE1, was produced in Escherichia coli and shown to convert not only 3-carboxy-cis,cis-muconate but also 3-sulfomuconate. ArCMLE1 was purified as a His-tagged enzyme variant, and the basic catalytic constants for the conversion of 3-carboxy-cis,cis-muconate and 3-sulfomuconate were determined. In contrast, Agrobacterium tumefaciens 3-carboxy-cis,cis-muconate lactonizing enzyme 1 could not, despite 87% sequence identity to ArCMLE1, use 3-sulfomuconate as substrate. The crystal structure of ArCMLE1 was determined at 2.2 A resolution. Consistent with the sequence, it showed that the C-terminal domain, present in all other members of the fumarase II family, is missing in ArCMLE1. Nonetheless, both the tertiary and quaternary structures, and the structure of the active site, are similar to those of Pseudomonas putida 3-carboxy-cis,cis-muconate lactonizing enzyme. One principal difference is that ArCMLE1 contains an Arg, as opposed to a Trp, in the active site. This indicates that activation of the carboxylic nucleophile by a hydrophobic environment is not required for lactonization, unlike earlier proposals [Yang J, Wang Y, Woolridge EM, Arora V, Petsko GA, Kozarich JW & Ringe D (2004) Biochemistry43, 10424-10434]. We identified citrate and isocitrate as noncompetitive inhibitors of ArCMLE1, and found a potential binding pocket for them on the enzyme outside the active site.

Factor H (FH) is the key regulator of the alternative pathway of complement. The carboxyl-terminal domains 19-20 of FH interact with the major opsonin C3b, glycosaminoglycans, and endothelial cells. Mutations within this area are associated with atypical haemolytic uremic syndrome (aHUS), a disease characterized by damage to endothelial cells, erythrocytes, and kidney glomeruli. The structure of recombinant FH19-20, solved at 1.8 A by X-ray crystallography, reveals that the short consensus repeat domain 20 contains, unusually, a short alpha-helix, and a patch of basic residues at its base. Most aHUS-associated mutations either destabilize the structure or cluster in a unique region on the surface of FH20. This region is close to, but distinct from, the primary heparin-binding patch of basic residues. By mutating five residues in this region, we show that it is involved, not in heparin, but in C3b binding. Therefore, the majority of the aHUS-associated mutations on the surface of FH19-20 interfere with the interaction between FH and C3b. This obviously leads to impaired control of complement attack on plasma-exposed cell surfaces in aHUS.

Family II pyrophosphatases (PPases), recently found in bacteria and archaebacteria, are Mn(2+)-containing metalloenzymes with two metal-binding subsites (M1 and M2) in the active site. These PPases can use a number of other divalent metal ions as the cofactor but are inactive with Zn(2+), which is known to be a good cofactor for family I PPases. We report here that the Mg(2+)-bound form of the family II PPase from Streptococcus gordonii is nearly instantly activated by incubation with equimolar Zn(2+), but the activity thereafter decays on a time scale of minutes. The activation of the Mn(2+)-form by Zn(2+) was slower but persisted for hours, whereas activation was not observed with the Ca(2+)- and apo-forms. The bound Zn(2+) could be removed from PPase by prolonged EDTA treatment, with a complete recovery of activity. On the basis of the effect of Zn(2+) on PPase dimerization, the Zn(2+) binding constant appeared to be as low as 10(-12) M for S. gordonii PPase. Similar effects of Zn(2+) and EDTA were observed with the Mg(2+)- and apo-forms of Streptococcus mutans and Bacillus subtilis PPases. The effects of Zn(2+) on the apo- and Mg(2+)-forms of HQ97 and DE15 B. subtilis PPase variants (modified M2 subsite) but not of HQ9 variant (modified M1 subsite) were similar to that for the Mn(2+)-form of wild-type PPase. These findings can be explained by assuming that (a) the PPase tightly binds Mg(2+) and Mn(2+) at the M2 subsite; (b) the activation of the corresponding holoenzymes by Zn(2+) results from its binding to the M1 subsite; and (c) the subsequent inactivation of Mg(2+)-PPase results from Zn(2+) migration to the M2 subsite. The inability of Zn(2+) to activate apo-PPase suggests that Zn(2+) binds more tightly to M2 than to M1, allowing direct binding to M2. Zn(2+) is thus an efficient cofactor at subsite M1 but not at subsite M2.

The crystal structure of the recombinant collagen-binding domain of Yersinia adhesin YadA from Yersinia enterocolitica serotype O:3 was solved at 1.55 A resolution. The trimeric structure is composed of head and neck regions, and the collagen binding head region is a novel nine-coiled left-handed parallel beta-roll. Before the beta-roll, the polypeptide loops from one monomer to the rest, and after the beta-roll the neck region does the same, making the transition from the globular head region to the narrower stalk domain. This creates an intrinsically stable 'lock nut' structure. The trimeric form of YadA is required for collagen binding, and mutagenesis of its surface residues allowed identification of a putative collagen-binding surface. Furthermore, a new structure-sequence motif for YadA beta-roll was used to identify putative YadA-head-like domains in a variety of human and plant pathogens. Such domains may therefore be a common bacterial strategy for avoiding host response.

Soluble inorganic pyrophosphatases (PPases) form two nonhomologous families, denoted I and II, that have similar active-site structures but different catalytic activities and metal cofactor specificities. Family II PPases, which are often found in pathogenic bacteria, are more active than family I PPases, and their best cofactor is Mn(2+) rather than Mg(2+), the preferred cofactor of family I PPases. Here, we present results of a detailed kinetic analysis of a family II PPase from Streptococcus gordonii (sgPPase), which was undertaken to elucidate the factors underlying the different properties of family I and II PPases. We measured rates of PP(i) hydrolysis, PP(i) synthesis, and P(i)/water oxygen exchange catalyzed by sgPPase with Mn(2+), Mg(2+), or Co(2+) in the high-affinity metal-binding site and Mg(2+) in the other sites, as well as the binding affinities for several active-site ligands (metal cofactors, fluoride, and P(i)). On the basis of these data, we deduced a minimal four-step kinetic scheme and evaluated microscopic rate constants for all eight relevant reaction steps. Comparison of these results with those obtained previously for the well-known family I PPase from Saccharomyces cerevisiae (Y-PPase) led to the following conclusions: (a) catalysis by sgPPase does not involve the enzyme-PP(i) complex isomerization known to occur in family I PPases; (b) the values of k(cat) for the magnesium forms of sgPPase and Y-PPase are similar because of similar rates of bound PP(i) hydrolysis and product release; (c) the marked acceleration of sgPPase catalysis in the presence of Mn(2+) and Co(2+) results from a combined effect of these ions on bound PP(i) hydrolysis and P(i) release; (d) sgPPase exhibits lower affinity for both PP(i) and P(i); and (e) sgPPase and Y-PPase exhibit similar values of k(cat)/K(m), which characterizes the PPase efficiency in vivo (i.e., at nonsaturating PP(i) concentrations).

GafD in Escherichia coli G (F17) fimbriae is associated with diarrheal disease, and the structure of the ligand-binding domain, GafD1-178, has been determined at 1.7 A resolution in the presence of the receptor sugar N -acetyl- d -glucosamine. The overall fold is a β-barrel jelly-roll fold. The ligand-binding site was identified and localized to the side of the molecule. Receptor binding is mediated by side-chain as well main-chain interactions. Ala43-Asn44, Ser116-Thr117 form the sugar acetamide specificity pocket, while Asp88 confers tight binding and Trp109 appears to position the ligand. There is a disulfide bond that rigidifies the acetamide specificity pocket. The three fimbrial lectins, GafD, FimH and PapG share similar β-barrel folds but display different ligand-binding regions and disulfide-bond patterns. We suggest an evolutionary path for the evolution of the very diverse fimbrial lectins from a common ancestral fold.

The structure of inactive pyruvate formate-lyase in complex with a natural substrate, pyruvate, was solved at 2.7 A resolution. Both active sites of the homodimeric enzyme are occupied by pyruvate; additional binding sites were not found. Pyruvate was found in a cleft close to the active-site cysteines 418 and 419, with the carboxyl group in contact with arginines 176 and 435 and the methyl group within van der Waals distance of Phe327. It is believed that the binding site of pyruvate is not the position of pyruvate as the reaction initiates, as conformational changes occur during activation of the enzyme.

Yeast (Saccharomyces cerevisiae) pyrophosphatase (Y-PPase) is a tight homodimer with two active sites separated in space from the subunit interface. The present study addresses the effects of mutation of four amino acid residues at the subunit interface on dimer stability and catalytic activity. The W52S variant of Y-PPase is monomeric up to an enzyme concentration of 300 microm, whereas R51S, H87T, and W279S variants produce monomer only in dilute solutions at pHor = 8.5, as revealed by sedimentation, gel electrophoresis, and activity measurements. Monomeric Y-PPase is considerably more sensitive to the SH reagents N-ethylmaleimide and p-hydroxymercurobenzosulfonate than the dimeric protein. Additionally, replacement of a single cysteine residue (Cys(83)), which is not part of the subunit interface or active site, with Ser resulted in insensitivity of the monomer to SH reagents and stabilization against spontaneous inactivation during storage. Active site ligands (Mg(2+) cofactor, P(i) product, and the PP(i) analog imidodiphosphate) stabilized the W279S dimer versus monomer predominantly by decreasing the rate of dimer to monomer conversion. The monomeric protein exhibited a markedly increased (5-9-fold) Michaelis constant, whereas k(cat) remained virtually unchanged, compared with dimer. These results indicate that dimerization of Y-PPase improves its substrate binding performance and, conversely, that active site adjustment through cofactor, product, or substrate binding strengthens intersubunit interactions. Both effects appear to be mediated by a conformational change involving the C-terminal segment that generally shields the Cys(83) residue in the dimer.

Background: It is unknown why patients with autoantibodies against complement factor H (CFH) lack homologous CFHR1 protein. Results: The autoantibody epitope on CFH was identified, and the structure of the corresponding part of CFHR1 was solved. Conclusion: The autoantigenic epitope of CFH and its homologous site in CFHR1 are structurally different. Significance: A plausible explanation for formation of autoantibodies due to CFHR1 deficiency in autoimmune atypical hemolytic uremic syndrome was obtained., Atypical hemolytic uremic syndrome (aHUS) is characterized by complement attack against host cells due to mutations in complement proteins or autoantibodies against complement factor H (CFH). It is unknown why nearly all patients with autoimmune aHUS lack CFHR1 (CFH-related protein-1). These patients have autoantibodies against CFH domains 19 and 20 (CFH19–20), which are nearly identical to CFHR1 domains 4 and 5 (CFHR14–5). Here, binding site mapping of autoantibodies from 17 patients using mutant CFH19–20 constructs revealed an autoantibody epitope cluster within a loop on domain 20, next to the two buried residues that are different in CFH19–20 and CFHR14–5. The crystal structure of CFHR14–5 revealed a difference in conformation of the autoantigenic loop in the C-terminal domains of CFH and CFHR1, explaining the variation in binding of autoantibodies from some aHUS patients to CFH19–20 and CFHR14–5. The autoantigenic loop on CFH seems to be generally flexible, as its conformation in previously published structures of CFH19–20 bound to the microbial protein OspE and a sialic acid glycan is somewhat altered. Cumulatively, our data suggest that association of CFHR1 deficiency with autoimmune aHUS could be due to the structural difference between CFHR1 and the autoantigenic CFH epitope, suggesting a novel explanation for CFHR1 deficiency in the pathogenesis of autoimmune aHUS.

Among the members of the fibroblast growth factor receptor family the FGFR4 has demonstrated strong dependence on heparin-like material for its activation by fibroblast growth factors. We have produced and characterized a recombinant human FGFR4 extracellular domain (FGFR4ed), in order to study its biochemical properties in isolated conditions. The FGFR4ed was expressed in an insect cell system and purified from the culture medium by Ni(2+)-affinity and gel filtration chromatography. Pure FGFR4ed was tested for FGF- and heparin-binding by covalent crosslinking experiments and by biosensor analysis. In solution, FGFR4ed formed complexes with acidic FGF (FGF-1) and basic FGF (FGF-2), both in the presence and absence of heparin. Immobilized FGFR4 also bound FGF-8 besides FGF-1 and FGF-2. Furthermore, heparin alone induced receptor oligomerization on the surface of the receptor coupled chip. Thus, the recombinant FGFR4ed revealed properties described for the cellular form of this receptor and can be used for interaction studies.

Human α2(C2)-adrenergic receptor was expressed in Escherichia coli as a fusion protein with Bacillus circulans var. alcalophilus cyclomaltodextrin glucanotransferase. For the determination of the expression level (0.6 mg of solubilized fusion protein l−1 of E. coli culture), a two-site immunometric assay based on two monoclonal antibodies with different epitopes was developed.

The objective is to generate milligram quantities of recombinant human α2C2 adrenergic receptor for X-ray crystallographic studies. It has been cloned in Saccharomyces cerevisiae, and the production level is at best about 13 pmol/mg of membrane protein, as estimated by radio-ligand binding assay. The receptor is solubilized with sucrose monolaurate followed by immunoaffinity purification and reconstitution into phospholipid vesicles. The efficiency of solubilization and immuno-purification are 60% and 91%, respectively.

A homohexameric molecule of Escherichia coli pyrophosphatase is arranged as a dimer of trimers, with an active site present in each of its six monomers. Earlier we reported that substitution of His136 and His140 in the intertrimeric subunit interface splits the molecule into active trimers (Velichko, I. S., Mikalahti, K., Kasho, V. N., Dudarenkov, V. Y., Hyytia, T., Goldman, A., Cooperman, B. S., Lahti, R., and Baykov, A. A. (1998) Biochemistry 37, 734–740). Here we demonstrate that additional substitutions of Tyr77 and Gln80 in the intratrimeric interface give rise to moderately active dimers or virtually inactive monomers, depending on pH, temperature, and Mg2+ concentration. Successive dissociation of the hexamer into trimers, dimers, and monomers progressively decreases the catalytic efficiency (by 106-fold in total), and conversion of a trimer into dimer decreases the affinity of one of the essential Mg2+-binding sites/monomer. Disruptive substitutions predominantly in the intratrimeric interface stabilize the intertrimeric interface and vice versa, suggesting that the optimal intratrimeric interaction is not compatible with the optimal intertrimeric interaction. Because of the resulting “conformational strain,” hexameric wild-type structure appears to be preformed to bind substrate. A hexameric triple variant substituted at Tyr77, Gln80, and His136 exhibits positive cooperativity in catalysis, consistent with this model.