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RNA-binding proteins (RBPs) are a major class of proteins that interact with RNAs to change their fate or function. RBPs and the ribonucleoprotein complexes they constitute are involved in many essential cellular processes. In many cases, the molecular details of RBP:RNA interactions differ between viruses, prokaryotes and eukaryotes, making prokaryotic and viral RBPs good potential drug targets. However, targeting RBPs with small molecules has so far been met with limited success as RNA-binding sites tend to be extended, shallow and dynamic with a mixture of charged, polar and hydrophobic interactions. Here, we show that peptide nucleic acids (PNAs) with nucleic acid-like binding properties and a highly stable peptide-like backbone can be used to target some RBPs. We have designed PNAs to mimic the short RNA stem-loop sequence required for the initiation of prokaryotic signal recognition particle (SRP) assembly, a target for antibiotics development. Using a range of biophysical and biochemical assays, the designed PNAs were demonstrated to fold into a hairpin structure, bind the targeted protein and compete with the native RNA hairpin to inhibit SRP formation. To show the applicability of PNAs against other RBPs, a PNA was also shown to bind Nsp9 from SARS-CoV-2, a protein that exhibits non-sequence-specific RNA binding but preferentially binds hairpin structures. Taken together, our results support that PNAs can be a promising class of compounds for targeting RNA-binding activities in RBPs., Competing Interests: NO authors have competing interests., (Copyright: © 2024 Zhong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Encephalopathies are brain dysfunctions that lead to cognitive, sensory, and motor development impairments. Recently, the identification of several mutations within the N -methyl-D-aspartate receptor (NMDAR) have been identified as significant in the etiology of this group of conditions. However, a complete understanding of the underlying molecular mechanism and changes to the receptor due to these mutations has been elusive. We studied the molecular mechanisms by which one of the first mutations within the NMDAR GluN1 ligand binding domain, Ser688Tyr, causes encephalopathies. We performed molecular docking, randomly seeded molecular dynamics simulations, and binding free energy calculations to determine the behavior of the two major co-agonists: glycine and D-serine, in both the wild-type and S688Y receptors. We observed that the Ser688Tyr mutation leads to the instability of both ligands within the ligand binding site due to structural changes associated with the mutation. The binding free energy for both ligands was significantly more unfavorable in the mutated receptor. These results explain previously observed in vitro electrophysiological data and provide detailed aspects of ligand association and its effects on receptor activity. Our study provides valuable insight into the consequences of mutations within the NMDAR GluN1 ligand binding domain.

Group A Streptococcus (GAS) is a strict human pathogen possessing a unique pathogenic trait that utilizes the cooperative activity of NAD + -glycohydrolase (NADase) and Streptolysin O (SLO) to enhance its virulence. How NADase interacts with SLO to synergistically promote GAS cytotoxicity and intracellular survival is a long-standing question. Here, the structure and dynamic nature of the NADase/SLO complex are elucidated by X-ray crystallography and small-angle scattering, illustrating atomic details of the complex interface and functionally relevant conformations. Structure-guided studies reveal a salt-bridge interaction between NADase and SLO is important to cytotoxicity and resistance to phagocytic killing during GAS infection. Furthermore, the biological significance of the NADase/SLO complex in GAS virulence is demonstrated in a murine infection model. Overall, this work delivers the structure-functional relationship of the NADase/SLO complex and pinpoints the key interacting residues that are central to the coordinated actions of NADase and SLO in the pathogenesis of GAS infection., (© 2023. The Author(s).)

Dynamic nuclear polarization NMR spectroscopy was used to investigate the effect of the antimicrobial peptide (AMP) maculatin 1.1 on E. coli cells. The enhanced 15 N NMR signals from nucleic acids, proteins and lipids identified a number of unanticipated physiological responses to peptide stress, revealing that membrane-active AMPs can have a multi-target impact on E. coli cells. DNP-enhanced 15 N-observed 31 P-dephased REDOR NMR allowed monitoring how Mac1 induced DNA condensation and prevented intermolecular salt bridges between the main E. coli lipid phosphatidylethanolamine (PE) molecules. The latter was supported by similar results obtained using E. coli PE lipid systems. Overall, the ability to monitor the action of antimicrobial peptides in situ will provide greater insight into their mode of action., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

In Gram-negative bacteria, the β-barrel assembly machinery (BAM) complex catalyses the assembly of β-barrel proteins into the outer membrane, and is composed of five subunits: BamA, BamB, BamC, BamD and BamE. Once assembled, - β-barrel proteins can be involved in various functions including uptake of nutrients, export of toxins and mediating host-pathogen interactions, but the precise mechanism by which these ubiquitous and often essential β-barrel proteins are assembled is yet to be established. In order to determine the relative positions of BAM subunits in the membrane environment we reconstituted each subunit into a biomimetic membrane, characterizing their interaction and structural changes by Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) and neutron reflectometry. Our results suggested that the binding of BamE, or a BamDE dimer, to BamA induced conformational changes in the polypeptide transported-associated (POTRA) domains of BamA, but that BamB or BamD alone did not promote any such changes. As monitored by neutron reflectometry, addition of an unfolded substrate protein extended the length of POTRA domains further away from the membrane interface as part of the mechanism whereby the substrate protein was folded into the membrane., (Copyright © 2021. Published by Elsevier B.V.)

Glaucoma, a major cause of irreversible blindness worldwide, is associated with elevated intraocular pressure (IOP) and progressive loss of retinal ganglion cells (RGCs) that undergo apoptosis. A mechanism for RGCs injury involves impairment of neurotrophic support and exogenous supply of neurotrophic factors has been shown to be beneficial. However, neurotrophic factors can have widespread effects on neuronal tissues, thus targeting neurotrophic support to injured neurons may be a better neuroprotective strategy. In this study, we have encapsulated LM22A-4, a small neurotrophic factor mimetic, into Annexin V-conjugated cubosomes (L4-ACs) for targeted delivery to injured RGCs in a model of acute IOP elevation, which is induced by acute IOP elevation. We have tested cubosomes formulations that encapsulate from 9% to 33% LM22A-4. Our data indicated that cubosomes encapsulating 9% and 17% LM22A-4 exhibited a mixture of Pn3m/Im3m cubic phase, whereas 23% and 33% showed a pure Im3m cubic phase. We found that 17% L4-ACs with Pn3m/Im3m symmetries showed better in-situ and in-vitro lipid membrane interactions than the 23% and 33% L4-ACs with Im3m symmetry. In vivo experiments showed that 17% L4-ACs targeted the posterior retina and the optic nerve head, which prevented RGCs loss and improved functional outcomes in a mouse model of acute IOP elevation. These results provide evidence that Annexin V-conjugated cubosomes-based LM22A-4 delivery may be a useful targeted approach to prevent the progression of RGCs loss in glaucoma. STATEMENT OF SIGNIFICANCE: Recent studies suggest that the therapy of effectively delivering neurotrophic factors to the injured retinal ganglion cells (RGCs) could promote the survival of RGCs in glaucoma. Our present work has for the first time used cubosomes as an active targeted delivery system and have successfully delivered a neuroprotective drug to the damaged RGCs in vivo. Our new cubosomal formulation can protect apoptotic cell death in vitro and in vivo, showing that cubosomes are a promising drug carrier system for ocular drug delivery and glaucoma treatment. We have further found that by controlling cubosomes in Pn3m phase we can facilitate delivery of neuroprotective drug through apoptotic membranes. This data, we believe, has important implications for future design and formulation of cubosomes for therapeutic applications., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

Malignant pleural mesothelioma is an aggressive cancer with poor prognosis. Here we have investigated in vitro efficacy of BAMLET and BLAGLET complexes (anti-cancer complexes consisting of oleic acid and bovine α-lactalbumin or β-lactoglobulin respectively) in killing mesothelioma cells, determined BAMLET and BLAGLET structures, and investigated possible biological mechanisms. We performed cell viability assays on 16 mesothelioma cell lines. BAMLET and BLAGLET having increasing oleic acid content inhibited human and rat mesothelioma cell line proliferation at decreasing doses. Most of the non-cancer primary human fibroblasts were more resistant to BAMLET than were human mesothelioma cells. BAMLET showed similar cytotoxicity to cisplatin-resistant, pemetrexed-resistant, vinorelbine-resistant, and parental rat mesothelioma cells, indicating the BAMLET anti-cancer mechanism may be different to drugs currently used to treat mesothelioma. Cisplatin, pemetrexed, gemcitabine, vinorelbine, and BAMLET, did not demonstrate a therapeutic window for mesothelioma compared with immortalised non-cancer mesothelial cells. We demonstrated by quantitative PCR that ATP synthase is downregulated in mesothelioma cells in response to regular dosing with BAMLET. We sought structural insight for BAMLET and BLAGLET activity by performing small angle X-ray scattering, circular dichroism, and scanning electron microscopy. Our results indicate the structural mechanism by which BAMLET and BLAGLET achieve increased cytotoxicity by holding increasing amounts of oleic acid in an active cytotoxic state encapsulated in increasingly unfolded protein. Our structural studies revealed similarity in the molecular structure of the protein components of these two complexes and in their encapsulation of the fatty acid, and differences in the microscopic structure and structural stability. BAMLET forms rounded aggregates and BLAGLET forms long fibre-like aggregates whose aggregation is more stable than that of BAMLET due to intermolecular disulphide bonds. The results reported here indicate that BAMLET and BLAGLET may be effective second-line treatment options for mesothelioma., Competing Interests: WBC is associated with the following patent application relating to small peptides and related compounds in the treatment of human disease [Entitled: Method of screening for inhibitors of sPLA2-IIA, International Patent Application PCT/AU2011/000781]. GR is an inventor on a patent (US patent 9006200) owned by the Asbestos Diseases Research Foundation involving a microRNA-based approach for treating malignant pleural mesothelioma. All other authors declare no potential competing financial, non-financial, professional, or personal interests relating to this manuscript. These disclosures do not alter our adherence to all the PLOS ONE policies on sharing data and materials.

Correct disulfide bond formation is essential for proper folding of many proteins, including bacterial virulence factors. The suppressor of copper sensitivity (Scs) proteins have roles in dithiol/disulfide interchange and the bacterial response to copper stress. Encoded in a four-gene cassette (ScsABCD) present in many Gram-negative bacteria, the Scs proteins are enigmatic and poorly characterized. Here, we show that the periplasmic α-domain of the membrane protein ScsB in the Gram-negative bacterium Proteus mirabilis forms a redox relay with the soluble periplasmic protein PmScsC. We also found that the periplasmic α-domain is sufficient to activate the disulfide isomerase activity of PmScsC. The crystal structure of PmScsBα at a resolution of 1.54 Å revealed that it comprises two structurally similar immunoglobulin-like folds, one of which includes a putative redox-active site with the sequence C XXX C. We confirmed the importance of these cysteine residues for PmScsBα function, and in addition, we engineered cysteine variants that produced a stable complex between PmScsC and PmScsBα. Using small-angle X-ray and neutron scattering analyses with contrast variation, we determined a low-resolution structure of the PmScsC-PmScsBα complex. The structural model of this complex suggested that PmScsBα uses both of its immunoglobulin-like folds to interact with PmScsC and revealed that the highly dynamic PmScsC becomes ordered upon PmScsBα binding. These findings add to our understanding of the poorly characterized Scs proteins., (© 2018 Furlong et al.)

The protein microenvironment surrounding the flavin cofactor in flavoenzymes is key to the efficiency and diversity of reactions catalysed by this class of enzymes. X-ray diffraction structures of oxidoreductase flavoenzymes have revealed recurrent features which facilitate catalysis, such as a hydrogen bond between a main chain nitrogen atom and the flavin redox center (N5). A neutron diffraction study of cholesterol oxidase has revealed an unusual elongated main chain nitrogen to hydrogen bond distance positioning the hydrogen atom towards the flavin N5 reactive center. Investigation of the structural features which could cause such an unusual occurrence revealed a positively charged lysine side chain, conserved in other flavin mediated oxidoreductases, in a second shell away from the FAD cofactor acting to polarize the peptide bond through interaction with the carbonyl oxygen atom. Double-hybrid density functional theory calculations confirm that this electrostatic arrangement affects the N-H bond length in the region of the flavin reactive center. We propose a novel second-order partial-charge interaction network which enables the correct orientation of the hydride receiving orbital of N5. The implications of these observations for flavin mediated redox chemistry are discussed.

Large protein complexes assemble spontaneously, yet their subunits do not prematurely form unwanted aggregates. This paradox is epitomized in the bacterial flagellar motor, a sophisticated rotary motor and sensory switch consisting of hundreds of subunits. Here we demonstrate that Escherichia coli FliG, one of the earliest-assembling flagellar motor proteins, forms ordered ring structures via domain-swap polymerization, which in other proteins has been associated with uncontrolled and deleterious protein aggregation. Solution structural data, in combination with in vivo biochemical cross-linking experiments and evolutionary covariance analysis, revealed that FliG exists predominantly as a monomer in solution but only as domain-swapped polymers in assembled flagellar motors. We propose a general structural and thermodynamic model for self-assembly, in which a structural template controls assembly and shapes polymer formation into rings.

Mechanisms for transcription factor recognition of specific DNA base sequences are well characterized and recent studies demonstrate that the shape of these cognate binding sites is also important. Here, we uncover a new mechanism where the transcription factor GabR simultaneously recognizes two cognate binding sites and the shape of a 29 bp DNA sequence that bridges these sites. Small-angle X-ray scattering and multi-angle laser light scattering are consistent with a model where the DNA undergoes a conformational change to bend around GabR during binding. In silico predictions suggest that the bridging DNA sequence is likely to be bendable in one direction and kinetic analysis of mutant DNA sequences with biolayer interferometry, allowed the independent quantification of the relative contribution of DNA base and shape recognition in the GabR-DNA interaction. These indicate that the two cognate binding sites as well as the bendability of the DNA sequence in between these sites are required to form a stable complex. The mechanism of GabR-DNA interaction provides an example where the correct shape of DNA, at a clearly distinct location from the cognate binding site, is required for transcription factor binding and has implications for bioinformatics searches for novel binding sites., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The genomes uncoupled 4 (GUN4) protein stimulates chlorophyll biosynthesis by increasing the activity of Mg-chelatase, the enzyme that inserts magnesium into protoporphyrin IX (PPIX) in the chlorophyll biosynthesis pathway. One of the roles of GUN4 is in binding PPIX and Mg-PPIX. In eukaryotes, GUN4 also participates in plastid-to-nucleus signalling, although the mechanism for this is unclear. Here, the first crystal structure of a eukaryotic GUN4, from Chlamydomonas reinhardtii, is presented. The structure is in broad agreement with those of previously solved cyanobacterial structures. Most interestingly, conformational divergence is restricted to several loops which cover the porphyrin-binding cleft. The conformational dynamics suggested by this ensemble of structures lend support to the understanding of how GUN4 binds PPIX or Mg-PPIX.

Mitochondrial dysfunction, due to mutations of the gene encoding succinate dehydrogenase (SDH), has been implicated in the development of adrenal phaeochromocytomas, sympathetic and parasympathetic paragangliomas, renal cell carcinomas, gastrointestinal stromal tumours and more recently pituitary tumours. Underlying mechanisms behind germline SDH subunit B (SDHB) mutations and their associated risk of disease are not clear. To investigate genotype-phenotype correlation of SDH subunit B (SDHB) variants, a homology model for human SDH was developed from a crystallographic structure. SDHB mutations were mapped, and biochemical effects of these mutations were predicted in silico. Results of structural modelling indicated that many mutations within SDHB are predicted to cause either failure of functional SDHB expression (p.Arg27*, p.Arg90*, c.88delC and c.311delAinsGG), or disruption of the electron path (p.Cys101Tyr, p.Pro197Arg and p.Arg242His). GFP-tagged WT SDHB and mutant SDHB constructs were transfected (HEK293) to determine biological outcomes of these mutants in vitro. According to in silico predictions, specific SDHB mutations resulted in impaired mitochondrial localisation and/or SDH enzymatic activity. These results indicated strong genotype-functional correlation for SDHB variants. This study reveals new insights into the effects of SDHB mutations and the power of structural modelling in predicting biological consequences. We predict that our functional assessment of SDHB mutations will serve to better define specific consequences for SDH activity as well as to provide a much needed assay to distinguish pathogenic mutations from benign variants., (© 2015 Society for Endocrinology.)

The HAMLET family of compounds (Human Alpha-lactalbumin Made Lethal to Tumours) was discovered during studies on the properties of human milk, and is a class of protein-lipid complexes having broad spectrum anti-cancer, and some specific anti-bacterial properties. The structure of HAMLET-like compounds consists of an aggregation of partially unfolded protein making up the majority of the compound's mass, with fatty acid molecules bound in the hydrophobic core. This is a novel protein-lipid structure and has only recently been derived by small-angle X-ray scattering analysis. The structure is the basis of a novel cytotoxicity mechanism responsible for anti-cancer activity to all of the around 50 different cancer cell types for which the HAMLET family has been trialled. Multiple cytotoxic mechanisms have been hypothesised for the HAMLET-like compounds, but it is not yet clear which of those are the initiating cytotoxic mechanism(s) and which are subsequent activities triggered by the initiating mechanism(s). In addition to the studies into the structure of these compounds, this review presents the state of knowledge of the anti-cancer aspects of HAMLET-like compounds, the HAMLET-induced cytotoxic activities to cancer and non-cancer cells, and the several prospective cell membrane and intracellular targets of the HAMLET family. The emerging picture is that HAMLET-like compounds initiate their cytotoxic effects on what may be a cancer-specific target in the cell membrane that has yet to be identified. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

In this work we have formulated Annexin V (ANX) decorated phosphatidylserine containing phytantriol (PSPhy) cubosomes to act as probes for the enhanced detection of apoptotic membranes in both model and in vitro cell systems. Small angle X-ray scattering (SAXS) and cryogenic-transmission electron microscopy (Cryo-TEM) indicated that ANX-containing PSPhy (ANX-PSPhy) cubosomes retain the Pn3m cubic symmetry and cubic phase nanoparticle characteristics of PSPhy cubosomes. The interaction of ANX-PSPhy cubosomes with apoptotic model and cellular membranes was also investigated using both quartz crystal microbalance with dissipation and confocal microscopy which confirmed that ANX-PSPhy cubosomes can selectively bind to apoptotic cells and model membranes. Neutron reflectometry has also been used to show strong binding of ANX-PSPhy cubosomes to a model apoptotic membrane, and in addition reveals changes in both the bilayer structure and in the internal structure of the cubosome in a region adjacent to the membrane as a result of material exchange. This material exchange between cubosome and apoptotic model bilayer was further demonstrated using Cryo-TEM. We have demonstrated that lipid bound protein, in this case Annexin V, can be used to target cubosome systems to biological surfaces in vitro., (© 2013 Elsevier Ltd. All rights reserved.)

Human group IIA secreted phospholipase A2 (hGIIA) promotes tumor growth and inflammation and can act independently of its well described catalytic lipase activity via an alternative poorly understood signaling pathway. With six chemically diverse inhibitors we show that it is possible to selectively inhibit hGIIA signaling over catalysis, and x-ray crystal structures illustrate that signaling involves a pharmacologically distinct surface to the catalytic site. We demonstrate in rheumatoid fibroblast-like synoviocytes that non-catalytic signaling is associated with rapid internalization of the enzyme and colocalization with vimentin. Trafficking of exogenous hGIIA was monitored with immunofluorescence studies, which revealed that vimentin localization is disrupted by inhibitors of signaling that belong to a rare class of small molecule inhibitors that modulate protein-protein interactions. This study provides structural and pharmacological evidence for an association between vimentin, hGIIA, and arachidonic acid metabolism in synovial inflammation, avenues for selective interrogation of hGIIA signaling, and new strategies for therapeutic hGIIA inhibitor design.

Calmodulin (CaM) expression is upregulated upon HIV-1 infection and interacts with proteins involved in viral processing, including the multifunctional HIV-1 MA protein. We present here the results of studies utilizing small-angle neutron scattering with contrast variation that, when considered in the light of earlier fluorescence and NMR data, show CaM binds MA in an extended open-clamp conformation via interactions with two tryptophans that are widely spaced in sequence and space. The interaction requires a disruption of the MA tertiary fold such that MA becomes highly extended in a long snakelike conformation. The CaM-MA interface is extensive, covering ~70% of the length of the MA such that regions known to be important in MA interactions with critical binding partners would be impacted. The CaM conformation is semiextended and as such is distinct from the classical CaM-collapse about short α-helical targets. NMR data show that upon dissociation of the CaM-MA complex, either by the removal of Ca 2+ or increasing ionic strength, MA reforms its native tertiary contacts. Thus, we observe a high level of structural plasticity in MA that may facilitate regulation of its activities via intracellular Ca 2+ -signaling during viral processing., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

When nerve cells communicate, vesicles from one neuron fuse with the presynaptic membrane releasing chemicals that signal to the next. Similarly, when insulin binds its receptor on adipocytes or muscle, glucose transporter-4 vesicles fuse with the cell membrane, allowing glucose to be imported. These essential processes require the interaction of SNARE proteins on vesicle and cell membranes, as well as the enigmatic protein Munc18 that binds the SNARE protein Syntaxin. Here, we show that in solution the neuronal protein Syntaxin1a interacts with Munc18-1 whether or not the Syntaxin1a N-peptide is present. Conversely, the adipocyte protein Syntaxin4 does not bind its partner Munc18c unless the N-peptide is present. Solution-scattering data for the Munc18-1:Syntaxin1a complex in the absence of the N-peptide indicates that this complex adopts the inhibitory closed binding mode, exemplified by a crystal structure of the complex. However, when the N-peptide is present, the solution-scattering data indicate both Syntaxin1a and Syntaxin4 adopt extended conformations in complexes with their respective Munc18 partners. The low-resolution solution structure of the open Munc18:Syntaxin binding mode was modeled using data from cross-linking/mass spectrometry, small-angle X-ray scattering, and small-angle neutron scattering with contrast variation, indicating significant differences in Munc18:Syntaxin interactions compared with the closed binding mode. Overall, our results indicate that the neuronal Munc18-1:Syntaxin1a proteins can adopt two alternate and functionally distinct binding modes, closed and open, depending on the presence of the N-peptide, whereas Munc18c:Syntaxin4 adopts only the open binding mode.

Complexes of Arthrobacter globiformis amine oxidase (AGAO) with the inhibitors benzylhydrazine and tranylcypromine (an antidepressant drug) have been refined at 1.86 and 1.65 A resolution, respectively. Both inhibitors form covalent adducts with the TPQ cofactor. A tyrosine residue, proposed to act as a gate to the AGAO active site, is in its open conformation.

The structure of a newly crystallized form of the copper-dependent amine oxidase from pea seedlings has been refined at a resolution of 2.2 A to a final R factor of 0.181. The structure (form II) was originally discovered during a study of xenon binding to copper-dependent amine oxidases as a probe for dioxygen-binding sites [Duff et al. (2004), J. Mol. Biol. 344, 599-607]. The form II crystals belong to space group P2(1), with two dimers in the asymmetric unit. The overall structure is very similar to the crystals of form I in space group P2(1)2(1)2(1) with a dimer in the asymmetric unit [Kumar et al. (1996), Structure, 4, 943-955]. In form I the last three residues (644-647) observable in the two subunits were apparently splayed apart. It was noted that the absence of a disulfide bond between the Cys647 residues of the two subunits was inconsistent with chemical evidence for the absence of free sulfhydryl groups. In both of the crystallographically independent dimers of form II the two subunits are clearly joined by a disulfide bridge between the C-terminal cysteine residues. This is only possible if the two polypeptide chains in the dimer adopt different conformations near the C-terminus so that the twofold symmetry is lost. A proline residue (645) two residues before the cysteine has a cis conformation in one chain and a trans conformation in the other. As a result, the disulfide bond lies more than 5 A from the twofold axis. The loss of local twofold symmetry in form II can be explained by intermolecular contacts, which provide an asymmetric environment.

Copper-containing amine oxidases are found in all the major kingdoms of life. They catalyse the oxidation of organic amines in the presence of molecular dioxygen to aldehydes and hydrogen peroxide. The catalytic centres contain a Cu atom and a topaquinone cofactor formed autocatalytically from a tyrosine residue in the presence of Cu and molecular oxygen. The structure of the Cu-containing amine oxidase from Arthrobacter globiformis, which was previously refined at 1.8 A resolution in space group C2 with unit-cell parameters a = 157.84, b = 63.24, c = 91.98 A, beta = 112.0 degrees [Wilce et al. (1997), Biochemistry, 36, 16116-16133], has been re-refined with newly recorded data at 1.55 A resolution. The structure has also been solved and refined at 2.2 A resolution in a new crystal form, space group C2, with unit-cell parameters a = 158.04, b = 64.06, c = 69.69 A, beta = 111.7 degrees.

Molecular wires comprising a Ru(II)- or Re(I)-complex head group, an aromatic tail group, and an alkane linker reversibly inhibit the activity of the copper amine oxidase from Arthrobacter globiformis (AGAO), with K(i) values between 6 muM and 37 nM. In the crystal structure of a Ru(II)-wire:AGAO conjugate, the wire occupies the AGAO active-site substrate access channel, the trihydroxyphenylalanine quinone cofactor is ordered in the "off-Cu" position with its reactive carbonyl oriented toward the inhibitor, and the "gate" residue, Tyr-296, is in the "open" position. Head groups, tail-group substituents, and linker lengths all influence wire-binding interactions with the enzyme.