Type IVa pili are ubiquitous and versatile bacterial cell surface filaments that undergo cycles of extension, adhesion and retraction powered by the cell-envelope spanning type IVa pilus machine (T4aPM). The overall architecture of the T4aPM and the location of 10 conserved core proteins within this architecture have been elucidated. Here, using genetics, cell biology, proteomics and cryo-electron tomography, we demonstrate that the PilY1 protein and four minor pilins, which are widely conserved in T4aP systems, are essential for pilus extension in Myxococcus xanthus and form a complex that is an integral part of the T4aPM. Moreover, these proteins are part of the extended pilus. Our data support a model whereby the PilY1/minor pilin complex functions as a priming complex in T4aPM for pilus extension, a tip complex in the extended pilus for adhesion, and a cork for terminating retraction to maintain a priming complex for the next round of extension.

Fig. 1. Peroxidase mechanism. (A) Traditional "water-modified" mechanism of CmpI formation. In this mechanism, peroxide first coordinates with the heme iron, followed by proton transfer to the distal peroxide O atom via an ordered water molecule and the distal His. The protonation state of the distal His depends on the His pK a . Our computational experiments indicate that the pK a substantially increases in CmpI. (B) Modified mechanism based on the observation that His52 in CCP CmpI is protonated in the neutron diffraction structure (8). Going from the initial peroxide complex to CmpI proceeds via two possible routes, one of which involves the Fe(IV)-OH intermediate. (C) Mechanism of CmpII reduction, which includes a protoncoupled electron transfer event resulting in a net transfer of a proton from the distal His to the ferryl O atom.

Since high levels of nitric oxide (NO) are implicated in neurodegenerative disorders, inhibition of the neuronal isoform of nitric oxide synthase (nNOS) and reduction of NO levels are therapeutically desirable. Nonetheless, many nNOS inhibitors mimic l-arginine and are poorly bioavailable. 2-Aminoquinoline-based scaffolds were designed with the hope that they could (a) mimic aminopyridines as potent, isoform-selective arginine isosteres and (b) possess chemical properties more conducive to oral bioavailability and CNS penetration. A series of these compounds was synthesized and assayed against purified nNOS enzymes, endothelial NOS (eNOS), and inducible NOS (iNOS). Several compounds built on a 7-substituted 2-aminoquinoline core are potent and isoform-selective; X-ray crystallography indicates that aminoquinolines exert inhibitory effects by mimicking substrate interactions with the conserved active site glutamate residue. The most potent and selective compounds, 7 and 15, were tested in a Caco-2 assay and showed good permeability and low efflux, suggesting high potential for oral bioavailability.

Mammals produce three isoforms of nitric oxide synthase (NOS): neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS). The overproduction of NO by nNOS is associated with a number of neurodegenerative disorders; therefore, a desirable therapeutic goal is the design of drugs that target nNOS but not the other isoforms. Crystallography, coupled with computational approaches and medicinal chemistry, has played a critical role in developing highly selective nNOS inhibitors that exhibit exceptional neuroprotective properties. For historic reasons, crystallography has focused on rat nNOS and bovine eNOS because these were available in high quality; thus, their structures have been used in structure-activity-relationship studies. Although these constitutive NOSs share more than 90% sequence identity across mammalian species for each NOS isoform, inhibitor-binding studies revealed that subtle differences near the heme active site in the same NOS isoform across species still impact enzyme-inhibitor interactions. Therefore, structures of the human constitutive NOSs are indispensible. Here, the first structure of human neuronal NOS at 2.03 Å resolution is reported and a different crystal form of human endothelial NOS is reported at 1.73 Å resolution.

The Bacteroidetes type IX secretion system (T9SS) consists of at least 19 components that translocate proteins with a type A or type B C-terminal domain (CTD) signal across the outer membrane. The overall organisation and architecture of this system including how the secretion pore (Sov) interacts with the other components is unknown. We used cryo-electron tomography to obtain the first images of the T9SS including PorK/N rings inside intact Porphyromonas gingivalis cells. Using proteomics, we identified a novel complex between Sov, PorV and PorA and showed that Sov interacts with the PorK/N rings via PorW and a new component PGN_1783. A separate complex comprising the outer membrane β barrel protein PorP, PorE, and the type B CTD protein PG1035 was also identified. Similarly, the Flavobacterium johnsoniae PorP-like protein, SprF was found bound to the major gliding motility adhesin, SprB. Based on these data, we propose cell surface anchorage for type B CTD proteins to PorP-like proteins and a unique model where the PorK/N rings function as an outer membrane barrier to maintain the close proximity of the translocon to the shuttle and 2 attachment complexes inside the rings, ensuring the harmonized secretion and cell surface attachment of the T9SS substrates.

Selective inhibition of neuronal nitric oxide synthase (nNOS) is an important therapeutic approach to target neurodegenerative disorders. However, the majority of the nNOS inhibitors developed are arginine mimetics and, therefore, suffer from poor bioavailability. We designed a novel strategy to combine a more pharmacokinetically favorable 2-imidazolylpyrimidine head with promising structural components from previous inhibitors. In conjunction with extensive structure–activity studies, several highly potent and selective inhibitors of nNOS were discovered. X-ray crystallographic analysis reveals that these type II inhibitors utilize the same hydrophobic pocket to gain strong inhibitory potency (13), as well as high isoform selectivity. Interestingly, select compounds from this series (9) showed good permeability and low efflux in a Caco-2 assay, suggesting potential oral bioavailability, and exhibited minimal off-target binding to 50 central nervous system receptors. Furthermore, even with heme-coordinating groups in the molecule, modifying other pharmacophoric fragments minimized undesirable inhibition of cytochrome P450s from human liver microsomes.

Using a new Titan Krios stage equipped with a single-axis holder, we developed two methods to accelerate the collection of tilt-series. We demonstrate a continuous-tilting method that can record a tilt-series in seconds, but with loss of details finer than ~4 nm. We also demonstrate a fastincremental method that can record a tilt-series several-fold faster than current methods and with similar resolution. We characterize the utility of both methods in real biological electron cryotomography workflows. We identify opportunities for further improvements in hardware and software and speculate on the impact such advances could have on structural biology. Highlights Continuous-tilting: acquire tilt-series in seconds  Currently limited to an estimated 4 nm resolution  Fast-incremental: acquire tilt-series several-fold faster than current methods  Can produce high quality subtomogram average at a fraction of the time

Neuronal nitric oxide synthase (nNOS) inhibition is a promising strategy to treat neurodegenerative disorders, but development of nNOS inhibitors is often hindered by poor pharmacokinetics. We previously developed a class of membrane-permeable 2-aminoquinoline inhibitors and later rearranged the scaffold to decrease off-target binding. However, the resulting compounds had decreased permeability, low human nNOS activity, and low selectivity versus human eNOS. In this study, 5-substituted phenyl ether-linked aminoquinolines and derivatives were synthesized and assayed against purified NOS isoforms. 5-Cyano compounds are especially potent and selective rat and human nNOS inhibitors. Activity and selectivity are mediated by the binding of the cyano group to a new auxiliary pocket in nNOS. Potency was enhanced by methylation of the quinoline and by introduction of simple chiral moieties, resulting in a combination of hydrophobic and auxiliary pocket effects that yielded high (∼500-fold) n/e selectivity. Importantly, Caco-2 assay also revealed improved membrane permeability over previous compounds.