A general strategy is described for the de novo design of proteins. In this strategy the sequence locations of hydrophobic and hydrophilic residues were specified explicitly, but the precise identities of the side chains were not constrained and varied extensively. This strategy was tested by constructing a large collection of synthetic genes whose protein products were designed to fold into four-helix bundle proteins. Each gene encoded a different amino acid sequence, but all sequences shared the same pattern of polar and nonpolar residues. Characterization of the expressed proteins indicated that most of the designed sequences folded into compact alpha-helical structures. Thus, a simple binary code of polar and nonpolar residues arranged in the appropriate order can drive polypeptide chains to collapse into globular alpha-helical folds.

Endocannabinoids are lipid signaling molecules that regulate a wide range of mammalian behaviors, including pain, inflammation, and cognitive/emotional state. The endocannabinoid anandamide is principally degraded by the integral membrane enzyme fatty acid amide hydrolase (FAAH), and there is currently much interest in developing FAAH inhibitors to augment endocannabinoid signaling in vivo. Here we report the discovery and detailed characterization of a highly efficacious and selective FAAH inhibitor PF-3845. Mechanistic and structural studies confirm that PF-3845 is a covalent inhibitor that carbamylates FAAH's serine nucleophile. PF-3845 selectively inhibits FAAH in vivo as determined by activity-based protein profiling and raises brain anandamide levels for up to 24 hrs, resulting in profound cannabinoid receptor-dependent reductions in inflammatory pain. These data thus designate PF-3845 as a valuable pharmacological tool for in vivo characterization of the endocannabinoid system.

Autotaxin (ATX) or ecto-nucleotide pyrophosphatase/phosphodiesterase-2 (ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemo-attractant for many cell types. ATX-LPA signaling has roles in various pathologies including tumour progression and inflammation. However, the molecular basis of substrate recognition and catalysis, and the mechanism of interaction with target cells, has been elusive. Here we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We identify a hydrophobic lipid-binding pocket and map key residues required for catalysis and selection between nucleotide and phospholipid substrates. We show that ATX interacts with cell-surface integrins via its N-terminal somatomedin-B-like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling, and enable new approaches to target ATX with small-molecule therapeutics.

The structure of a synaptic intermediate of the site-specific recombinase gammadelta resolvase covalently linked through Ser10 to two cleaved duplex DNAs has been determined at 3.4 angstrom resolution. This resolvase, activated for recombination by mutations, forms a tetramer whose structure is substantially changed from that of a presynaptic complex between dimeric resolvase and the cleavage site DNA. Because the two cleaved DNA duplexes that are to be recombined lie on opposite sides of the core tetramer, large movements of both protein and DNA are required to achieve strand exchange. The two dimers linked to the DNAs that are to be recombined are held together by a flat interface. This may allow a 180 degrees rotation of one dimer relative to the other in order to reposition the DNA duplexes for strand exchange.

Replicative DNA polymerases (DNAPs) move along template DNA in a processive manner. The structural basis of the mechanism of translocation has been better studied in the A-family of polymerases than in the B-family of replicative polymerases. To address this issue, we have determined the X-ray crystal structures of phi29 DNAP, a member of the protein-primed subgroup of the B-family of polymerases, complexed with primer-template DNA in the presence or absence of the incoming nucleoside triphosphate, the pre-and post-translocated states, respectively. Comparison of these structures reveals a mechanism of translocation that appears to be facilitated by the coordinated movement of two conserved tyrosine residues into the insertion site. This differs from the mechanism employed by the A-family polymerases, in which a conserved tyrosine moves into the templating and insertion sites during the translocation step. Polymerases from the two families also interact with downstream single-stranded template DNA in very different ways.

Analysis of nucleotide binding induced conformational changes in the current and previous HslU structures suggests a protein unfolding-coupled translocation mechanism. In this mechanism, unfolded polypeptides are threaded through the aligned pores of the ATPase and peptidase and translocated into the peptidase central chamber.

The integral membrane enzyme fatty acid amide hydrolase (FAAH) hydrolyzes the endocannabinoid anandamide and related amidated signaling lipids. Genetic or pharmacological inactivation of FAAH produces analgesic, anxiolytic, and antiinflammatory phenotypes but not the undesirable side effects of direct cannabinoid receptor agonists, indicating that FAAH may be a promising therapeutic target. Structure-based inhibitor design has, however, been hampered by difficulties in expressing the human FAAH enzyme. Here, we address this problem by interconverting the active sites of rat and human FAAH using site-directed mutagenesis. The resulting humanized rat (h/r) FAAH protein exhibits the inhibitor sensitivity profiles of human FAAH but maintains the high-expression yield of the rat enzyme. We report a 2.75-Å crystal structure of h/rFAAH complexed with an inhibitor, N-phenyl-4-(quinolin-3-ylmethyl)piperidine-1-carboxamide (PF-750), that shows strong preference for human FAAH. This structure offers compelling insights to explain the species selectivity of FAAH inhibitors, which should guide future drug design programs.anandamide ͉ crystal structure ͉ endocannabinoid ͉ fatty acid amides ͉ hydrolase F atty acid amide hydrolase (FAAH) is an integral membrane enzyme that hydrolyzes the fatty acid amide class of lipid transmitters (1, 2). FAAH substrates include the endogenous cannabinoid N-arachidonoyl ethanolamine (anandamide) (3), the antiinflammatory factor N-palmitoyl ethanolamine (PEA) (4), the sleep-inducing substance 9(Z)-octadecenamide (oleamide) (5), and the satiating signal N-oleoyl ethanolamine (OEA) (6). FAAH inactivation by either chemical inhibition or genetic deletion of the FAAH gene leads to elevated endogenous levels of fatty acid amides and a range of behavioral effects that include analgesia (7-12), anxiolytic (8,13,14), antidepressant (13, 15), sleep-enhancing (16), and antiinflammatory (17-19) phenotypes. Importantly, these behavioral phenotypes occur in the absence of alterations in motility, weight gain, or body temperature that are typically observed with direct cannabinoid receptor 1 (CB1) agonists. Inhibition of FAAH thus may offer an attractive way to produce the therapeutically beneficial phenotypes of activating the endocannabinoid system without the undesirable side effects that are observed with direct CB1 agonists.FAAH is a member of a large class of enzymes termed the amidase signature class (20). These enzymes, which span all kingdoms of life, use an unusual Ser-Ser-Lys catalytic triad (21, 22) to hydrolyze amide bonds on a wide range of small-molecule substrates. Despite their atypical catalytic mechanism, amidase signature enzymes are inactivated by general classes of serine hydrolase inhibitors [e.g., trifluoromethyl ketones (23, 24), fluorophosphonates (25), ␣-ketoheterocycles (26), carbamates (8, 27)]. First-generation FAAH inhibitors, such as methyl arachidonyl fluorophosphonate (MAFP) (25), were substrate-derived in structure and therefore lack selectivity for FAAH relative to other l...

The observed nucleotide-dependent conformational changes in HslU and their governing principles provide a framework for the mechanistic understanding of other AAA(+) proteins.