Parallel Beta-strands Research Articles

Entropic Bristles Tune the Seeding Efficiency of Prion-Nucleating Fragments

SummaryPrions of lower eukaryotes are self-templating protein aggregates with cores formed by parallel in-register beta strands. Short aggregation-prone glutamine (Q)- and asparagine (N)-rich regions embedded in longer disordered domains have been proposed to act as nucleation sites that initiate refolding of soluble prion proteins into highly ordered fibrils, termed amyloid. We demonstrate that a short Q/N-rich peptide corresponding to a proposed nucleation site in the prototype Saccharomyces cerevisiae prion protein Sup35 is sufficient to induce infectious cytosolic prions in mouse neuroblastoma cells ectopically expressing the soluble Sup35 NM prion domain. Embedding this nucleating core in a non-native N-rich sequence that does not form amyloid but acts as an entropic bristle quadruples seeding efficiency. Our data suggest that large disordered sequences flanking an aggregation core in prion proteins act as not only solubilizers of the monomeric protein but also breakers of the formed amyloid fibrils, enhancing infectivity of the prion seeds.

The crystal structure of the Rv0301‐Rv0300 VapBC‐3 toxin—antitoxin complex from M. tuberculosis reveals a Mg2+ ion in the active site and a putative RNA‐binding site

VapBC pairs account for 45 out of 88 identified toxin-antitoxin (TA) pairs in the Mycobacterium tuberculosis (Mtb) H37Rv genome. A working model suggests that under times of stress, antitoxin molecules are degraded, releasing the toxins to slow the metabolism of the cell, which in the case of VapC toxins is via their RNase activity. Otherwise the TA pairs remain bound to their promoters, autoinhibiting transcription. The crystal structure of Rv0301-Rv0300, an Mtb VapBC TA complex determined at 1.49 Å resolution, suggests a mechanism for these three functions: RNase activity, its inhibition by antitoxin, and its ability to bind promoter DNA. The Rv0301 toxin consists of a core of five parallel beta strands flanked by alpha helices. Three proximal aspartates coordinate a Mg²⁺ ion forming the putative RNase active site. The Rv0300 antitoxin monomer is extended in structure, consisting of an N-terminal beta strand followed by four helices. The last two helices wrap around the toxin and terminate near the putative RNase active site, but with different conformations. In one conformation, the C-terminal arginine interferes with Mg²⁺ ion coordination, suggesting a mechanism by which the antitoxin can inhibit toxin activity. At the N-terminus of the antitoxin, two pairs of Ribbon-Helix-Helix (RHH) motifs are related by crystallographic twofold symmetry. The resulting hetero-octameric complex is similar to the FitAB system, but the two RHH motifs are about 30 Å closer together in the Rv0301-Rv0300 complex, suggesting either a different span of the DNA recognition sequence or a conformational change.

Crystal structure of the DNA-recognition component of the bacterial virus Sf6 genome-packaging machine

In herpesviruses and many bacterial viruses, genome-packaging is a precisely mediated process fulfilled by a virally encoded molecular machine called terminase that consists of two protein components: A DNA-recognition component that defines the specificity for packaged DNA, and a catalytic component that provides energy for the packaging reaction by hydrolyzing ATP. The terminase docks onto the portal protein complex embedded in a single vertex of a preformed viral protein shell called procapsid, and pumps the viral DNA into the procapsid through a conduit formed by the portal. Here we report the 1.65 A resolution structure of the DNA-recognition component gp1 of the Shigella bacteriophage Sf6 genome-packaging machine. The structure reveals a ring-like octamer formed by interweaved protein monomers with a highly extended fold, embracing a tunnel through which DNA may be translocated. The N-terminal DNA-binding domains form the peripheral appendages surrounding the octamer. The central domain contributes to oligomerization through interactions of bundled helices. The C-terminal domain forms a barrel with parallel beta-strands. The structure reveals a common scheme for oligomerization of terminase DNA-recognition components, and provides insights into the role of gp1 in formation of the packaging-competent terminase complex and assembly of the terminase with the portal, in which ring-like protein oligomers stack together to form a continuous channel for viral DNA translocation.

Heterologous Stacking of Prion Protein Peptides Reveals Structural Details of Fibrils and Facilitates Complete Inhibition of Fibril Growth

Fibrils play an important role in the pathogenesis of amyloidosis; however, the underlying mechanisms of the growth process and the structural details of fibrils are poorly understood. Crucial in the fibril formation of prion proteins is the stacking of PrP monomers. We previously proposed that the structure of the prion protein fibril may be similar as a parallel left-handed beta-helix. The beta-helix is composed of spiraling rungs of parallel beta-strands, and in the PrP model residues 105-143 of each PrP monomer can contribute two beta-helical rungs to the growing fibril. Here we report data to support this model. We show that two cyclized human PrP peptides corresponding to residues 105-124 and 125-143, based on two single rungs of the left-handed beta-helical core of the human PrP(Sc) fibril, show spontaneous cooperative fibril growth in vitro by heterologous stacking. Because the structural model must have predictive value, peptides were designed based on the structure rules of the left-handed beta-helical fold that could stack with prion protein peptides to stimulate or to block fibril growth. The stimulator peptide was designed as an optimal left-handed beta-helical fold that can serve as a template for fibril growth initiation. The inhibiting peptide was designed to bind to the exposed rung but frustrate the propagation of the fibril growth. The single inhibitory peptide hardly shows inhibition, but the combination of the inhibitory with the stimulatory peptide showed complete inhibition of the fibril growth of peptide huPrP-(106-126). Moreover, the unique strategy based on stimulatory and inhibitory peptides seems a powerful new approach to study amyloidogenic fibril structures in general and could prove useful for the development of therapeutics.

De Novo Design of a βαβ Motif

A designer monomeric protein with a beta alpha beta fold--two parallel beta strands connected by an alpha helix (see structure)--was constructed solely from coded amino acids. The high thermal stability of the structure is due to a large extent to tryptophan-tryptophan interactions between the two beta strands.

BETASCAN: Probable β-amyloids Identified by Pairwise Probabilistic Analysis

Amyloids and prion proteins are clinically and biologically important β-structures, whose supersecondary structures are difficult to determine by standard experimental or computational means. In addition, significant conformational heterogeneity is known or suspected to exist in many amyloid fibrils. Recent work has indicated the utility of pairwise probabilistic statistics in β-structure prediction. We develop here a new strategy for β-structure prediction, emphasizing the determination of β-strands and pairs of β-strands as fundamental units of β-structure. Our program, BETASCAN, calculates likelihood scores for potential β-strands and strand-pairs based on correlations observed in parallel β-sheets. The program then determines the strands and pairs with the greatest local likelihood for all of the sequence's potential β-structures. BETASCAN suggests multiple alternate folding patterns and assigns relative a priori probabilities based solely on amino acid sequence, probability tables, and pre-chosen parameters. The algorithm compares favorably with the results of previous algorithms (BETAPRO, PASTA, SALSA, TANGO, and Zyggregator) in β-structure prediction and amyloid propensity prediction. Accurate prediction is demonstrated for experimentally determined amyloid β-structures, for a set of known β-aggregates, and for the parallel β-strands of β-helices, amyloid-like globular proteins. BETASCAN is able both to detect β-strands with higher sensitivity and to detect the edges of β-strands in a richly β-like sequence. For two proteins (Aβ and Het-s), there exist multiple sets of experimental data implying contradictory structures; BETASCAN is able to detect each competing structure as a potential structure variant. The ability to correlate multiple alternate β-structures to experiment opens the possibility of computational investigation of prion strains and structural heterogeneity of amyloid. BETASCAN is publicly accessible on the Web at http://betascan.csail.mit.edu.

Structural diversity of the soluble trimers of the human amylin(20–29) peptide revealed by molecular dynamics simulations

The human islet amyloid polypeptide (hIAPP) or amylin is a 37-residue hormone found as amyloid deposits in pancreatic extracts of nearly all type 2 diabetes patients. The fragment 20-29 of sequence SNNFGAILSS (hIAPP20-29) has been shown to be responsible for the amyloidogenic propensities of the full length protein. Various polymorphic forms of hIAPP20-29 fibrils were described by using Fourier transform infrared (FTIR) and solid-state NMR experiments: unseeded hIAPP20-29 fibril with out-of-register antiparallel beta-strands, and two forms of seeded hIAPP20-29 fibril, with in-register antiparallel or in-register parallel beta-strands. As a first step toward understanding this polymorphism, we explore the equilibrium structures of the soluble hIAPP20-29 trimer, using multiple molecular dynamics (MD) simulations with the Optimized Potential for Efficient structure Prediction (OPEP) coarse-grained implicit solvent force field for a total length of 3.2 micros. Although, the trimer is found mainly random coil, consistent with the signal measured experimentally during the lag phase of hIAPP20-29 fibril formation, the central FGAIL residues have a relative high propensity to form interpeptide beta-sheets and antiparallel beta-strands are more probable than parallel beta-strands. One MD-predicted out-of-register antiparallel three-stranded beta-sheet matches exactly the FTIR-derived unseeded hIAPP20-29 fibril model. Our simulations, however, do not reveal any evidence of in-register parallel or in-register antiparallel beta-sheets as reported for seeded hIAPP20-29 fibrils. All these results indicate that fibril polymorphism is partially encoded in a trimer.

Synthesis of Diacetylene‐Containing Peptide Building Blocks and Amphiphiles, Their Self‐Assembly and Topochemical Polymerization in Organic Solvents

A series of functional iodoacetylenes was prepared and converted into the corresponding diacetylene-substituted amino acids and peptides via Pd/Cu-promoted sp-sp carbon cross-coupling reactions. The unsymmetrically substituted diacetylenes can be incorporated into oligopeptides without a change in the oligopeptide strand's directionality. Thus, a series of oligopeptide-based, amphiphilic diacetylene model compounds was synthesized, and their self-organization as well as their UV-induced topochemical polymerizability was investigated in comparison to related polymer-substituted macromonomers. Solution-phase IR spectroscopy, gelation experiments, and UV spectroscopy helped to confirm that a minimum of five N-H...O=C hydrogen-bonding sites was required in order to obtain reliable aggregation into stable beta-sheet-type secondary structures in organic solvents. Furthermore, the non-equidistant spacing of these hydrogen-bonding sites was proven to invariably lead to beta-sheets with a parallel beta-strand orientation, and the characteristic IR-spectroscopic signatures of the latter in organic solution was identified. Scanning force micrographs of the organogels revealed that compounds with six hydrogen-bonding sites gave rise to high aspect ratio nanoscopic fibrils with helical superstructures but, in contrast to the related macromonomers, did not lead to uniform supramolecular polymers. The UV-induced topochemical polymerization within the beta-sheet aggregates was successful, proving parallel beta-strand orientation and highlighting the effect of the number and pattern of N-H...O=C hydrogen-bonding sites as well as the hydrophobic residue in the molecular structure on the formation of higher structures and reactivity.

Structural Insights into the Polymorphism of Amyloid-Like Fibrils Formed by Region 20−29 of Amylin Revealed by Solid-State NMR and X-ray Fiber Diffraction

Many unrelated proteins and peptides can assemble into amyloid or amyloid-like nanostructures, all of which share the cross-beta motif of repeat arrays of beta-strands hydrogen-bonded along the fibril axis. Yet, paradoxically, structurally polymorphic fibrils may derive from the same initial polypeptide sequence. Here, solid-state nuclear magnetic resonance (SSNMR) analysis of amyloid-like fibrils of the peptide hIAPP 20-29, corresponding to the region S (20)NNFGAILSS (29) of the human islet amyloid polypeptide amylin, reveals that the peptide assembles into two amyloid-like forms, (1) and (2), which have distinct structures at the molecular level. Rotational resonance SSNMR measurements of (13)C dipolar couplings between backbone F23 and I26 of hIAPP 20-29 fibrils are consistent with form (1) having parallel beta-strands and form (2) having antiparallel strands within the beta-sheet layers of the protofilament units. Seeding hIAPP 20-29 with structurally homogeneous fibrils from a 30-residue amylin fragment (hIAPP 8-37) produces morphologically homogeneous fibrils with similar NMR properties to form (1). A model for the architecture of the seeded fibrils is presented, based on the analysis of X-ray fiber diffraction data, combined with an extensive range of SSNMR constraints including chemical shifts, torsional angles, and interatomic distances. The model features a cross-beta spine comprising two beta-sheets with an interface defined by residues F23, A25, and L27, which form a hydrophobic zipper. We suggest that the energies of formation for fibril form containing antiparallel and parallel beta-strands are similar when both configurations can be stabilized by a core of hydrophobic contacts, which has implications for the relationship between amino acid sequence and amyloid polymorphism in general.

β2-Microglobulin Amyloid Fragment Organization and Morphology and Its Comparison to Aβ Suggests That Amyloid Aggregation Pathways Are Sequence Specific

Beta2-microglobulin (beta2-m) can form dialysis-related amyloid deposits. The structure of a fragment of beta2-m (K3, Ser20-Lys41) in the oligomeric state has recently been solved. We modeled equilibrium structures of K3 oligomers with different organizations (single and double layers) and morphologies (linear-like and annular-like) for the wild-type and mutants using all-atom molecular dynamics (MD) simulations. We focused on the sheet-to-sheet association force, which is the key in the amyloid organization and morphology. For the linear-like morphology, we observed two stable organizations: (i) single-layered parallel-stranded beta-sheets and (ii) double-layered parallel-stranded antiparallel beta-sheets stacked perpendicular to the fibril axis through the hydrophobic N-terminal-N-terminal (NN) interface. No stable annular structures were observed. The structural instability of the annular morphology was mainly attributed to electrostatic repulsion of three negatively charged residues (Asp15, Glu17, and Asp19) projecting from the same beta-strand surface. Linear-like and annular-like double-layered oligomers with the NN interface are energetically more favorable than other oligomers with C-terminal-C-terminal (CC) or C-terminal-N-terminal (CN) interfaces, emphasizing the importance of hydrophobic interactions and side-chain packing in stabilizing these oligomers. Moreover, only linear-like structures, rather than annular structures, with parallel beta-strands and antiparallel beta-sheet arrangements are possible intermediate states for the K3 beta2-m amyloid fibrils in solution. Comparing the beta2-m fragment with Abeta indicates that while both adopt similar beta-strand-turn-beta-strand motifs, the final amyloid structures can be dramatically different in size, structure, and morphology due to differences in side-chain packing arrangements, intermolecular driving forces, sequence composition, and residue positions, suggesting that the mechanism leading to distinct morphologies and the aggregation pathways is sequence specific.

Shikimate Kinase: A Potential Target for Development of Novel Antitubercular Agents

Tuberculosis (TB) remains the leading cause of mortality due to a bacterial pathogen, Mycobacterium tuberculosis. However, no new classes of drugs for TB have been developed in the past 30 years. Therefore there is an urgent need to develop faster acting and effective new antitubercular agents, preferably belonging to new structural classes, to better combat TB, including MDR-TB, to shorten the duration of current treatment to improve patient compliance, and to provide effective treatment of latent tuberculosis infection. The enzymes in the shikimate pathway are potential targets for development of a new generation of antitubercular drugs. The shikimate pathway has been shown by disruption of aroK gene to be essential for the Mycobacterium tuberculosis. The shikimate kinase (SK) catalyses the phosphorylation of the 3-hydroxyl group of shikimic acid (shikimate) using ATP as a co-substrate. SK belongs to family of nucleoside monophosphate (NMP) kinases. The enzyme is an alpha/beta protein consisting of a central sheet of five parallel beta-strands flanked by alpha-helices. The shikimate kinases are composed of three domains: Core domain, Lid domain and Shikimate-binding domain. The Lid and Shikimate-binding domains are responsible for large conformational changes during catalysis. More recently, the precise interactions between SK and substrate have been elucidated, showing the binding of shikimate with three charged residues conserved among the SK sequences. The elucidation of interactions between MtSK and their substrates is crucial for the development of a new generation of drugs against tuberculosis through rational drug design.

A Membrane-proximal Tetracysteine Motif Contributes to Assembly of CD3δϵ and CD3γϵ Dimers with the T Cell Receptor

Assembly of the T cell receptor (TCR) with its dimeric signaling modules, CD3deltaepsilon, CD3gammaepsilon, and zetazeta, is organized by transmembrane (TM) interactions. Each of the three assembly steps requires formation of a three-helix interface involving one particular basic TCR TM residue and two acidic TM residues of the respective signaling dimer. The extracellular domains of CD3deltaepsilon and CD3gammaepsilon contribute to assembly, but TCR interaction sites on CD3 dimers have not been defined. The structures of the extracellular domains of CD3deltaepsilon and CD3gammaepsilon demonstrated parallel beta-strands ending at the first cysteine in the CXXCXEXXX motif present in the stalk segment of each CD3 chain. Mutation of the membrane-proximal cysteines impaired assembly of either CD3 dimer with TCR, and little complex was isolated when all four membrane-proximal cysteines were mutated to alanine. These mutations had, however, no discernable effect on CD3deltaepsilon or CD3gammaepsilon dimerization. CD3deltaepsilon assembled with a TCRalpha mutant that lacked both immunoglobulin domains, but shortening of the TCRalpha connecting peptide reduced assembly, consistent with membrane-proximal TCRalpha-CD3deltaepsilon interactions. Chelation of divalent cations did not affect assembly, indicating that coordination of a cation by the tetracysteine motif was not required. The membrane-proximal cysteines were within close proximity but only formed covalent CD3 dimers when one cysteine was mutated. The four cysteines may thus form two intrachain disulfide bonds integral to the secondary structure of CD3 stalk regions. The three-chain interaction theme first established for the TM domains thus extends into the membrane-proximal domains of TCRalpha-CD3deltaepsilon and TCRbeta-CD3gammaepsilon.

NMR solution structure of the acylphosphatase from Escherichia coli

The solution structure of Escherichia coli acylphosphatase (E. coli AcP), a small enzyme catalyzing the hydrolysis of acylphosphates, was determined by (1)H and (15)N NMR and restrained modelling calculation. In analogy with the other members of AcP family, E. coli AcP shows an alpha/beta sandwich domain composed of four antiparallel and one parallel beta-strand, assembled in a five-stranded beta-sheet facing two antiparallel alpha-helices. The pairwise RMSD values calculated for the backbone atoms of E. coli and Sulfolobus solfataricus AcP, Bovine common type AcP and Horse muscle AcP are 2.18, 5.31 and 5.12 A, respectively. No significant differences are present in the active site region and the catalytic residue side chains are consistently positioned in the structures.

How Well Does Poisson−Boltzmann Implicit Solvent Agree with Explicit Solvent? A Quantitative Analysis

We have quantitatively studied the performance of a finite-difference Poisson-Boltzmann implicit solvent with respect to the TIP3P explicit solvent in a range of systems of biochemical interest. An overall agreement was found between the tested implicit and explicit solvents for hydrogen-bonding/salt-bridging dimers and peptide monomers and dimers of different conformations and different lengths. These comparative analyses also indicate a good transferability of empirically optimized parameters for the implicit solvent from small training molecules to large testing peptides. However, deviations between the two tested solvents are also apparent. Specifically, a consistent deviation was observed when hydrogen-bonding or salt-bridging dimers are within 4-6 A. The deviation reaches a maximum at about 5.5 A, the so-called water-bridging distance. The tested implicit solvent, even with optimized parameters, cannot capture the subtle fluctuation in the distance-dependent reaction field energy profiles, although smoothed profiles can still be obtained and are in overall agreement with those in the explicit solvent. Interestingly, the same mechanism underlining the above discrepancy is also responsible for the larger deviations of certain peptide conformations, such as parallel beta-strand dimers. It is likely that the observed discrepancy may cause improper conformational distributions in simulations with the implicit solvent when hydrogen-bonding or salt-bridging interactions are crucial, such as secondary structure populations in proteins. Validation of the implicit solvent with optimized parameters in dynamics simulations will be the next step to study the influences of the observed discrepancy at biological conditions.

Side‐chain entropy effects on protein secondary structure formation

Loss of conformational entropy is one of the primary factors opposing protein folding. Both the backbone and side-chain of each residue in a protein will have their freedom of motion restricted in the final folded structure. The type of secondary structure of which a residue is part will have a significant impact on how much side-chain entropy is lost. Side-chain conformational entropies have previously been determined for folded proteins, simple models of unfolded proteins, alpha-helices, and a dipeptide model for beta-strands, but not for polyproline II (PII) helices. In this work, we present side-chain conformational estimates for the three regular secondary structure types: alpha-helices, beta-strands, and PII helices. Entropies are estimated from Monte Carlo computer simulations. Beta-strands are modeled as two structures, parallel and antiparallel beta-strands. Our data indicate that restraining a residue to the PII helix or antiparallel beta-strand conformations results in side-chain entropies equal to or higher than those obtained by restraining residues to the parallel beta-strand conformation. Side-chains in the alpha-helix conformation have the lowest side-chain entropies. The observation that extended structures retain the most side-chain entropy suggests that such structures would be entropically favored in unfolded proteins under folding conditions. Our data indicate that the PII helix conformation would be somewhat favored over beta-strand conformations, with antiparallel beta-strand favored over parallel. Notably, our data imply that, under some circumstances, residues may gain side-chain entropy upon folding. Implications of our findings for protein folding and unfolded states are discussed.

The 1.49 Å Resolution Crystal Structure of PsbQ from Photosystem II of Spinacia oleracea Reveals a PPII Structure in the N-terminal Region

We report the high-resolution structure of the spinach PsbQ protein, one of the main extrinsic proteins of higher plant photosystem II (PSII). The crystal structure shows that there are two well-defined regions in PsbQ, the C-terminal region (residues 46-149) folded as a four helix up-down bundle and the N-terminal region (residues 1-45) that is loosely packed. This structure provides, for the first time, insights into the crucial N-terminal region. First, two parallel beta-strands cross spatially, joining the beginning and the end of the N-terminal region of PsbQ. Secondly, the residues Pro9-Pro10-Pro11-Pro12 form a left-handed helix (or a polyproline type II (PPII) structure), which is stabilized by hydrogen bonds between the Pro peptide carbonyl groups and solvent water molecules. Thirdly, residues 14-33 are not visible in the electron density map, suggesting that this loop might be very flexible and presumably extended when PsbQ is free in solution. On the basis of the essential role of the N-terminal region of PsbQ in binding to PSII, we propose that both the PPII structure and the missing loop are key secondary structure elements in the recognition of specific protein-protein interactions between PsbQ and other oxygen-evolving complex extrinsic and/or intrinsic proteins of PSII. In addition, the PsbQ crystal coordinates two zinc ions, one of them is proposed to have a physiological role in higher plants, on the basis of the full conservation of the ligand protein residues in the sequence subfamily.

Polyproline II helix is the preferred conformation for unfolded polyalanine in water.

Does aqueous solvent discriminate among peptide conformers? To address this question, we computed the solvation free energy of a blocked, 12-residue polyalanyl-peptide in explicit water and analyzed its solvent structure. The peptide was modeled in each of 4 conformers: alpha-helix, antiparallel beta-strand, parallel beta-strand, and polyproline II helix (P(II)). Monte Carlo simulations in the canonical ensemble were performed at 300 K using the CHARMM 22 forcefield with TIP3P water. The simulations indicate that the solvation free energy of P(II) is favored over that of other conformers for reasons that defy conventional explanation. Specifically, in these 4 conformers, an almost perfect correlation is found between a residue's solvent-accessible surface area and the volume of its first solvent shell, but neither quantity is correlated with the observed differences in solvation free energy. Instead, solvation free energy tracks with the interaction energy between the peptide and its first-shell water. An additional, previously unrecognized contribution involves the conformation-dependent perturbation of first-shell solvent organization. Unlike P(II), beta-strands induce formation of entropically disfavored peptide:water bridges that order vicinal water in a manner reminiscent of the hydrophobic effect. The use of explicit water allows us to capture and characterize these dynamic water bridges that form and dissolve during our simulations.

TOPS: an enhanced database of protein structural topology.

The TOPS database holds topological descriptions of protein structures. These compact and highly abstract descriptions reduce the protein fold to a sequence of Secondary Structure Elements (SSEs) and three sets of pairwise relationships between them, hydrogen bonds relating parallel and anti- parallel beta strands, spatial adjacencies relating neighbouring SSEs, and the chiralities of selected supersecondary structures, including connections in betaalphabeta units and between parallel alpha helices. The database is used as a resource for visualizing folding topologies, fast topological pattern searching and structure comparison. Here, significant enhancements to the TOPS database are described. The topological description has been enhanced to include packing relationships between helices, which significantly improves the description of protein folds with little beta strand content. Further, the topological description has been annotated with sequence information. The query interfaces to the database have been improved and the new version can be found at http://www.tops.leeds.ac.uk/.

Crystal structure of the conserved protein TT1542 from Thermus thermophilus HB8.

The TT1542 protein from Thermus thermophilus HB8 is annotated as a conserved hypothetical protein, and belongs to the DUF158 family in the Pfam database. A BLAST search revealed that homologs of TT1542 are present in a wide range of organisms. The TT1542 homologs in eukaryotes, PIG-L in mammals, and GPI12 in yeast and protozoa, have N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) de-N-acetylase activity. Although most of the homologs in prokaryotes are hypothetical and have no known function, Rv1082 and Rv1170 from Mycobacterium tuberculosis are enzymes involved in the mycothiol detoxification pathway. Here we report the crystal structure of the TT1542 protein at 2.0 A resolution, which represents the first structure for this superfamily of proteins. The structure of the TT1542 monomer consists of a twisted beta-sheet composed of six parallel beta-strands and one antiparallel beta-strand (with the strand order 3-2-1-4-5-7-6) sandwiched between six alpha-helices. The N-terminal five beta-strands and four alpha-helices form an incomplete Rossmann fold-like structure. The structure shares some similarity to the sugar-processing enzymes with Rossmann fold-like domains, especially those of the GPGTF (glycogen phosphorylase/glycosyl transferase) superfamily, and also to the NAD(P)-binding Rossmann fold domains. TT1542 is a homohexamer in the crystal and in solution, the six monomers forming a cylindrical structure. Putative active sites are suggested by the structure and conserved amino acid residues.

Ligand Selectivity of Gonadotropin Receptors: ROLE OF THE β-STRANDS OF EXTRACELLULAR LEUCINE-RICH REPEATS 3 AND 6 OF THE HUMAN LUTEINIZING HORMONE RECEPTOR

The difference in hormone selectivity between the human follicle-stimulating hormone receptor (hFSH-R) and human luteinizing hormone/chorionic gonadotropin receptor (hLH-R) is determined by their approximately 350 amino acid-long N-terminal receptor exodomains that allow the mutually exclusive binding of human follicle-stimulating hormone (hFSH) and human luteinizing hormone (hLH) when these hormones are present in physiological concentrations. The exodomains of each of these receptors consist of a nine-leucine-rich repeat-containing subdomain (LRR subdomain) flanked by N- and C-terminal cysteine-rich subdomains. Chimeric receptors, in which the structural subdomains of the hFSH-R exodomain were substituted with those of the hLH-R, showed a similar high responsiveness to human chorionic gonadotropin (hCG) and hLH as long as they harbored the LRR subdomain of the hLH-R. In addition, these chimeric receptors showed no responsiveness to hFSH. The LRR subdomains of the gonadotropin receptor exodomains are predicted to adopt a horseshoe-like conformation, of which the hormone-binding concave surface is composed of nine parallel beta-strands. Receptors in which individual beta-strands of the hFSH-R were replaced with the corresponding hLH-R sequences revealed that hCG and hLH selectivity is predominantly determined by hLH-R beta-strands 3 and 6. A mutant receptor in which the hFSH-R beta-strands 3 and 6 were substituted simultaneously with their hLH-R counterparts displayed a responsiveness to hCG and hLH similar to that of the wild type hLH-R. Responsiveness to hFSH was not affected by most beta-strand substitutions, suggesting the involvement of multiple low-impact determinants for this hormone.