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The leukotriene B4 receptor 1 (BLT1) regulates the recruitment and chemotaxis of different cell types and plays a role in the pathophysiology of infectious, allergic, metabolic, and tumorigenic human diseases. Here we present a crystal structure of human BLT1 (hBLT1) in complex with a selective antagonist MK-D-046, developed for the treatment of type 2 diabetes and other inflammatory conditions. Comprehensive analysis of the structure and structure-activity relationship data, reinforced by site-directed mutagenesis and docking studies, reveals molecular determinants of ligand binding and selectivity toward different BLT receptor subtypes and across species. The structure helps to identify a putative membrane-buried ligand access channel as well as potential receptor binding modes of endogenous agonists. These structural insights of hBLT1 enrich our understanding of its ligand recognition and open up future avenues in structure-based drug design., Human leukotriene B4 receptors (BLT1 and BLT2) are members of the GPCR superfamily that respond to a potent pro-inflammatory lipid and chemoattractant LTB4. Here authors determined a crystal structure of the human BLT1 in complex with a selective antagonist MK-D-046 and provide insights into hBLT1 ligand recognition and its mechanism of action.

Single-domain antibody fragments known as VHH have emerged in the pharmaceutical industry as useful biotherapeutics. These molecules, which are naturally produced by camelids, share the characteristics of high affinity and specificity with traditional human immunoglobulins, while consisting of only a single heavy chain. Currently, the most common method for generating VHH is via animal immunization, which can be costly and time-consuming. Here we describe the development of a synthetic VHH library for in vitro selection of single domain binders. We combine structure-based design and next-generation sequencing analysis to build a library with characteristics that closely mimic the natural repertoire. To validate the performance of our synthetic library, we isolated VHH against three model antigens (soluble mouse PD-1 ectodomain, amyloid-β peptide, and MrgX1 GPCR) of different sizes and characteristics. We were able to isolate diverse binders targeting different epitopes with high affinity (as high as 5 nM) against all three targets. We then show that anti-mPD-1 binders have functional activity in a receptor blocking assay.

Glucagon is a 29-amino-acid peptide released from the α-cells of the islet of Langerhans, which has a key role in glucose homeostasis. Glucagon action is transduced by the class B G-protein-coupled glucagon receptor (GCGR), which is located on liver, kidney, intestinal smooth muscle, brain, adipose tissue, heart and pancreas cells, and this receptor has been considered an important drug target in the treatment of diabetes. Administration of recently identified small-molecule GCGR antagonists in patients with type 2 diabetes results in a substantial reduction of fasting and postprandial glucose concentrations. Although an X-ray structure of the transmembrane domain of the GCGR has previously been solved, the ligand (NNC0640) was not resolved. Here we report the 2.5 Å structure of human GCGR in complex with the antagonist MK-0893 (ref. 4), which is found to bind to an allosteric site outside the seven transmembrane (7TM) helical bundle in a position between TM6 and TM7 extending into the lipid bilayer. Mutagenesis of key residues identified in the X-ray structure confirms their role in the binding of MK-0893 to the receptor. The unexpected position of the binding site for MK-0893, which is structurally similar to other GCGR antagonists, suggests that glucagon activation of the receptor is prevented by restriction of the outward helical movement of TM6 required for G-protein coupling. Structural knowledge of class B receptors is limited, with only one other ligand-binding site defined--for the corticotropin-releasing hormone receptor 1 (CRF1R)--which was located deep within the 7TM bundle. We describe a completely novel allosteric binding site for class B receptors, providing an opportunity for structure-based drug design for this receptor class and furthering our understanding of the mechanisms of activation of these receptors.

Neurotransmitter sodium symporters are integral membrane proteins that remove chemical transmitters from the synapse and terminate neurotransmission mediated by serotonin, dopamine, noradrenaline, glycine and GABA (γ-aminobutyric acid). Crystal structures of the bacterial homologue, LeuT, in substrate-bound outward-occluded and competitive inhibitor-bound outward-facing states have advanced our mechanistic understanding of neurotransmitter sodium symporters but have left fundamental questions unanswered. Here we report crystal structures of LeuT mutants in complexes with conformation-specific antibody fragments in the outward-open and inward-open states. In the absence of substrate but in the presence of sodium the transporter is outward-open, illustrating how the binding of substrate closes the extracellular gate through local conformational changes: hinge-bending movements of the extracellular halves of transmembrane domains 1, 2 and 6, together with translation of extracellular loop 4. The inward-open conformation, by contrast, involves large-scale conformational changes, including a reorientation of transmembrane domains 1, 2, 5, 6 and 7, a marked hinge bending of transmembrane domain 1a and occlusion of the extracellular vestibule by extracellular loop 4. These changes close the extracellular gate, open an intracellular vestibule, and largely disrupt the two sodium sites, thus providing a mechanism by which ions and substrate are released to the cytoplasm. The new structures establish a structural framework for the mechanism of neurotransmitter sodium symporters and their modulation by therapeutic and illicit substances.

Neurotransmitter/sodium symporters (NSSs) couple the uptake of neurotransmitter with one or more sodium ions1–3, removing neurotransmitter from the synaptic cleft. NSSs are essential to the function of chemical synapses, are associated with multiple neurological diseases and disorders4, and are the targets of therapeutic and illicit drugs5. LeuT, a prokaryotic orthologue of the NSS family, is a model transporter for understanding the relationships between molecular mechanism and atomic structure in a broad range of sodium-dependent and sodium-independent secondary transporters6–13. At present there is a controversy over whether there are one or two high-affinity substrate binding sites in LeuT. The first-reported crystal structure of LeuT, together with subsequent functional and structural studies, provided direct evidence for a single, high-affinity, centrally located substrate-binding site, defined as the S1 site14,15. Recent binding, flux and molecular simulation studies, however, have been interpreted in terms of a model where there are two high-affinity binding sites: the central, S1, site and a second, the S2 site, located within the extracellular vestibule16. Furthermore, it was proposed that the S1 and S2 sites are allosterically coupled such that occupancy of the S2 site is required for the cytoplasmic release of substrate from the S1 site16. Here we address this controversy by performing direct measurement of substrate binding to wild-type LeuT and to S2 site mutants using isothermal titration calorimetry, equilibrium dialysis and scintillation proximity assays. In addition, we perform uptake experiments to determine whether the proposed allosteric coupling between the putative S2 site and the S1 site manifests itself in the kinetics of substrate flux. We conclude that LeuT harbours a single, centrally located, high-affinity substrate-binding site and that transport is well described by a simple, single-substrate kinetic mechanism.

Transmembrane sodium ion gradients provide energy that can be harnessed by so-called ‘secondary transporters’ to drive the translocation of solute molecules into a cell. Decades of intense study has proven the central role that sodium-coupled transporters play in many physiological processes, making them vital targets to treat many serious diseases. Within the last year, several sodium-coupled transporter crystal structures have been reported from different families, showing a remarkable structural conservation between functionally unrelated transporters. In this review we discuss these atomic resolution structures and how they illuminate the mechanistic principles of sodium-coupled transport of solutes across cellular membranes.

In this paper, we study restoration mechanisms to handle channel and link failures in an optical wavelength division multiplexed (WDM) wavelength-routed wide-area backbone network based on a mesh topology. The solution uses a small number of tunable lasers to provide restoration capability. We consider two types of failures: link failures and individual channel (or wavelength) failures that occur when one or more transceivers fail at a node that is the source of lightpath(s) or due to a failure in an intermediate node's optical switch fabric. We use the restoration mechanism that attempts to find alternate paths and resources after failure occurs. In our proposed mechanism, restoration is first attempted using the tunable lasers to transmit on the failed wavelengths. If all the failed lightpaths cannot be restored using the tunable lasers, unused wavelengths on the same link are used, if optical wavelength conversion is available. For the remaining lightpaths requiring restoration, two different link-level restoration mechanisms called redirection algorithm (RDA) and disjoint path algorithm (DPA) are used. Results based on discrete-event simulations to understand the performance of the proposed mechanisms, in terms of restoration efficiency and restoration times, are presented. The results show that for networks of varying size and node degree with 32 wavelengths on each link, using as few as eight tunable lasers per link provides good restoration efficiency under moderate traffic load. The performance of the proposed algorithms is compared to an earlier restoration mechanism based on broadcast, and it is seen that the proposed mechanism performs better, by offering both lower restoration times and higher restoration efficiency even with a small number of lasers. The impact of the number of tunable lasers on the performance is studied for failures occurring simultaneously on two links. It is seen that for a small number of such channel failures, as few as four tunable lasers per link are sufficient to recover from failures on a single link and on two links.

The ternary complex of Esche- richia coli adenylate kinase (ECAK) with its sub- strates adenosine monophosphate (AMP) and Mg- ATP, which catalyzes the reversible transfer of a phosphoryl group between adenosine triphosphate (ATP) and AMP, was studied using molecular dynam- ics. The starting structure for the simulation was assembled from the crystal structures of ECAK com- plexed with the bisubstrate analog diadenosine pen- taphosphate (AP5A) and of Bacillus stearothermophi- lus adenylate kinase complexed with AP5A, Mg 2 , and 4 coordinated water molecules, and by deleting 1 phosphate group from AP5A. The interactions of ECAK residues with the various moieties of ATP and AMP were compared to those inferred from NMR, X-ray crystallography, site-directed mutagenesis, and enzyme kinetic studies. The simulation sup- ports the hypothesis that hydrogen bonds between AMP's adenine and the protein are at the origin of the high nucleoside monophosphate (NMP) specific- ity of AK. The ATP adenine and ribose moieties are only loosely bound to the protein, while the ATP phosphates are strongly bound to surrounding resi- dues. The coordination sphere of Mg 2 , consisting of 4 waters and oxygens of the ATP - and -phos- phates, stays approximately octahedral during the simulation. The important role of the conserved Lys13 in the P loop in stabilizing the active site by bridging the ATP and AMP phosphates is evident. The influence of Mg 2 , of its coordination waters, and of surrounding charged residues in maintain- ing the geometry and distances of the AMP -phos- phate and ATP - and -phosphates is sufficient to support an associative reaction mechanism for phos- phoryl transfer. Proteins 2005;58:88 -100.

The adenylate kinase (AK) gene from Thermotoga neapolitana, a hyperthermophilic bacterium, was cloned and overexpressed in Escherichia coli, and the recombinant enzyme was biochemically characterized. The T. neapolitana AK (TNAK) sequence indicates that this enzyme belongs to the long bacterial AKs. TNAK contains the four cysteine residues that bind Zn(2+) in all Gram-positive AKs and in a few other Zn(2+)-containing bacterial AKs. Atomic emission spectroscopy and titration data indicate a content of 1 mol of Zn(2+)/mol of recombinant TNAK. The EDTA-treated enzyme has a melting temperature (T (m)=93.5 degrees C) 6.2 degrees C below that of the holoenzyme (99.7 degrees C), identifying Zn(2+) as a stabilizing feature in TNAK. TNAK is a monomeric enzyme with a molecular mass of approx. 25 kDa. TNAK displays V (max) and K (m) values at 30 degrees C identical with those of the E. coli AK at 30 degrees C, and displays very high activity at 80 degrees C, with a specific activity above 8000 units/mg. The unusually high activity of TNAK at 30 degrees C makes it an interesting model to test the role of enzyme flexibility in activity.

Backbone conformational dynamics of Thermotoga neapolitana adenylate kinase in the free form (TNAK) and inhibitor-bound form (TNAK*Ap5A) were investigated at 30 degrees C using (15)N NMR relaxation measurements and NMR monitored hydrogen-deuterium exchange. With kinetic parameters identical to those of Escherichia coli AK (ECAK) at 30 degrees C, TNAK is a unique hyperthermophilic enzyme. These catalytic properties make TNAK an interesting and novel model to study the interplay between protein rigidity, stability, and activity. Comparison of fast time scale dynamics (picosecond to nanosecond) in the open and closed states of TNAK and ECAK at 30 degrees C reveals a uniformly higher rigidity across all domains of TNAK. Within this framework of a rigid TNAK structure, several residues located in the AMP-binding domain and in the core-lid hinge regions display high picosecond to nanosecond time scale flexibility. Together with the recent comparison of ECAK dynamics with those of hyperthermophilic Aquifex aeolicus AK (AAAK), our results provide strong evidence for the role of picosecond to nanosecond time scale fluctuations in both stability and activity. In the slow time scales, TNAK's increased rigidity is not uniform but localized in the AMP-binding and lid domains. The core domain amides of ECAK and TNAK in the open and closed states show comparable protection against exchange. Significantly, the hinges framing the lid domain show similar exchange data in ECAK and TNAK open and closed forms. Our NMR relaxation and hydrogen-deuterium exchange studies therefore suggest that TNAK maintains high activity at 30 degrees C by localizing flexibility to the hinge regions that are key to facilitating conformational changes.

In this paper, we study restoration mechanisms to handle channel and link failures in an optical WDM wavelength-routed wide-area backbone network based on a mesh topology. The solution uses a small number of tunable lasers per link to provide restoration capability. In addition to link failures, we consider individual channel failures link-level mechanisms are presented: redirection algorithm (RDA) and disjoint path algorithm (DPA). These mechanisms use WDM-specific link information to compute the link restoration routes. We present results based on discrete-event simulations to understand the performance of the proposed mechanisms, in terms of restoration efficiency and restoration times. The results show that for networks of varying size and node degree with 32 wavelengths on each link, using as few as 8 tunable lasers per link provides good restoration efficiency under moderate traffic load. which might occur when one or more transceivers fail at the source of the lightpath or due to a failure in the switch fabric. Restoration is first attempted using the tunable lasers to transmit on the failed wavelengths. If all the failed lightpaths cannot be restored using the tunable lasers, unused wavelengths on the same link are used (this requires optical wavelength conversion at the nodes). For the remaining lightpaths requiring restoration, link-level restoration mechanisms are attempted. Two different© (2002) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.