Domain-swap polymerization drives the self-assembly of the bacterial flagellar motor.

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.

Binding of transcription factor GabR to DNA requires recognition of DNA shape at a location distinct from its cognate binding site.

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.

Solution structure studies of monomeric human TIP47/perilipin-3 reveal a highly extended conformation.

Tail-interacting protein of 47 kDa (TIP47) has two putative functions: lipid biogenesis and mannose 6-phosphate receptor recycling. Progress in understanding the molecular details of these two functions has been hampered by the lack of structural data on TIP47, with a crystal structure of the C-terminal domain of the mouse homolog constituting the only structural data in the literature so far. Our studies have first provided a strategy to obtain pure monodisperse preparations of the full-length TIP47/perilipin-3 protein, as well as a series of N-terminal truncation mutants with no exogenous sequences. These constructs have then enabled us to obtain the first structural characterization of the full-length protein in solution. Our work demonstrates that the N-terminal region of TIP47/perilipin-3, in contrast to the largely helical C-terminal region, is predominantly ß-structure with turns and bends. Moreover, we show that full-length TIP47/perilipin-3 adopts an extended conformation in solution, with considerable spatial separation of the N- and C-termini that would likely translate into a separation of functional domains.

Human cardiac myosin binding protein C: structural flexibility within an extended modular architecture.

New insights into the modular organization and flexibility of the N-terminal half of human cardiac myosin binding protein C (cMyBP-C) and information on the association state of the full-length protein have been deduced from a combined small-angle X-ray scattering (SAXS) and NMR study. SAXS data show that the first five immunoglobulin domains of cMyBP-C, which include those implicated in interactions with both myosin and actin, remain monodisperse and monomeric in solution and have a highly extended yet distinctively 'bent' modular arrangement that is similar to the giant elastic muscle protein titin. Analyses of the NMR and SAXS data indicate that a proline/alanine-rich linker connecting the cardiac-specific N-terminal C0 domain to the C1 domain provides significant structural flexibility at the N-terminus of the human isoform, while the modular arrangement of domains C1-C2-C3-C4 is relatively fixed. Domain fragments from the C-terminal half of the protein have a propensity to self-associate in vitro, while full-length bacterially expressed cMyBP-C forms flexible extended dimers at micromolar protein concentrations. In summary, our studies reveal that human cMyBP-C combines a distinctive modular architecture with regions of flexibility and that the N-terminal half of the protein is sufficiently extended to span the range of interfilament distances sampled within the dynamic environment of heart muscle. These structural features of cMyBP-C could facilitate its putative role as a molecular switch between actin and myosin and may contribute to modulating the transverse pliancy of the C-zone of the A-band across muscle sarcomeres.

A novel structure of an antikinase and its inhibitor.

In Bacillus subtilis, the KipI protein is a regulator of the phosphorelay governing the onset of sporulation. KipI binds the relevant sensor histidine kinase, KinA, and inhibits the autophosphorylation reaction. Gene homologues of kipI are found almost ubiquitously throughout the bacterial kingdom and are usually located adjacent to, and often fused with, kipA gene homologues. In B. subtilis, the KipA protein inhibits the antikinase activity of KipI thereby permitting sporulation. We have used a combination of biophysical techniques in order to understand the domain structure and shape of the KipI-KipA complex and probe the nature of the interaction. We also have solved the crystal structure of TTHA0988, a Thermus thermophilus protein of unknown function that is homologous to a KipI-KipA fusion. This structure, which is the first to be described for this class of proteins, provides unique insight into the nature of the KipI-KipA complex. The structure confirms that KipI and KipA are proteins with two domains, and the C-terminal domains belong to the cyclophilin family. These cyclophilin domains are positioned in the complex such that their conserved surfaces face each other to form a large "bicyclophilin" cleft. We discuss the sequence conservation and possible roles across species of this near-ubiquitous protein family, which is poorly understood in terms of function.

1H, 13C and 15N backbone and side chain resonance assignments of the N-terminal domain of the histidine kinase inhibitor KipI from Bacillus subtilis.

KipI is a sporulation inhibitor in Bacillus subtilis which acts by binding to the dimerisation and histidine phosphotransfer (DHp) domain of KinA, the principle input kinase in the phosphorelay responsible for sporulation. The (15)N, (13)C and (1)H chemical shift assignments of the N-terminal domain of KipI were determined using multidimensional, multinuclear NMR experiments. The N-terminal domain has two conformers and resonance assignments have been made for both conformers.

Identification of a stable flavin-thiolate adduct in heterotetrameric sarcosine oxidase.

Heterotetrameric sarcosine oxidase (TSOX) is a complex bifunctional flavoenzyme that contains two flavins. Most of the FMN in recombinant TSOX is present as a covalent adduct with an endogenous ligand. Enzyme denaturation disrupts the adduct, accompanied by release of a stoichiometric amount of sulfide. Enzyme containing>or=90% unmodified FMN is prepared by displacement of the endogenous ligand with sulfite, a less tightly bound competing ligand. Reaction of adduct-depleted TSOX with sodium sulfide produces a stable complex that resembles the endogenous TSOX adduct and known 4a-S-cysteinyl flavin adducts. The results provide definitive evidence for sulfide as the endogenous TSOX ligand and strongly suggest that the modified FMN is a 4a-sulfide adduct. A comparable reaction with sodium sulfide is not detected with other flavoprotein oxidases. A model of the postulated TSOX adduct suggests that it is stabilized by nearby residues that may be important in the electron transferase/oxidase function of the coenzyme.

Alteration in spectral properties on ligand binding reveals flexibility in monoamine oxidase.

Crystal structures have opened the door to understanding the mechanism and ligand specificities of MAO A and MAO B. We review here functional properties that suggest a flexibility in MAO that is likely to influence catalysis under different cellular conditions. The flexibility indicated by altered oxidation kinetics and a changed redox potential in the presence of a substrate was confirmed by circular dichroism spectroscopy. Circular dichroism also demonstrated alterations in the conformation of aromatic residues during reduction of MAO A and after covalent modification of the flavin. Visible spectra provide a convenient way to monitor ligand binding in the active site. Different groups near the flavin give different spectral changes. During reduction of MAO A, a distinct 412 nm peak appears after partial reduction. Recent work suggests that this may be a tyrosyl radical in equilibrium with the semiquinone of the flavin. Substrates prevent the appearance of the 412 nm peak but many inhibitors enhance it by preventing further reduction. We propose that steric effects in the active site could be the mechanism of this difference. Flexibility is also important for the transmission of the effects of modifying the surface thiols to the active site. Modification of multiple thiols results in inactivation but mutation of a single thiol, cysteine 374 in MAO A to alanine, decreased the catalytic potency (kcat/Km) by 30%. Thus, surface modification of MAO (for example, by oxidative stress) could reduce its activity.

A stable tyrosyl radical in monoamine oxidase A.

We present spectroscopic evidence consistent with the presence of a stable tyrosyl radical in partially reduced human monoamine oxidase (MAO) A. The radical forms following single electron donation to MAO A and exists in equilibrium with the FAD flavosemiquinone. Oxidative formation of the tyrosyl radical in MAO is not reliant on neighboring metal centers and uniquely requires reduction of the active site flavin to facilitate oxidation of a tyrosyl side chain. The identified tyrosyl radical provides the key missing link in support of the single electron transfer mechanism for amine oxidation by MAO enzymes.

Conformational changes in monoamine oxidase A in response to ligand binding or reduction.

The structure of monoamine oxidase B revealed three aromatic amino acid residues within contact distance of the flavin cofactor and a large number of aromatic residues in the substrate binding site. Circular dichroism (CD) spectroscopy can detect alterations in the environment of aromatic residues as a result of ligand binding or redox changes. CD spectra of MAO A indicate that a small inhibitor such d-amphetamine perturbs the aromatic residues very little, but binding of the larger pirlindole (2,3,3a,4,5,6-hexahydro-8-methyl-1H-pyrazino[3,2,1-j,k]carbazole hydrochloride) causes spectral changes consistent with the alteration of the environment of tyrosine and tryptophan residues in particular. Reduction of the flavin cofactor induces large enhancement of the CD signals in the aromatic region (260-310 nm). When covalent modification of the flavin by clorgyline accompanies reduction, the perturbation is even greater. In contrast to the static picture offered by crystallography, this study reveals changes in the aromatic cage on ligand binding and suggests that reduction of the cofactor substantially alters the environment of aromatic residues presumably near the flavin. In addition, the covalently modified reduced MAO A shows significant differences from the substrate-reduced enzyme.

Monoamine oxidase A inhibitory potency and flavin perturbation are influenced by different aspects of pirlindole inhibitor structure.

Reversible inhibitors of monoamine oxidase A (MAO A) are used as antidepressants. The influence of inhibitors such as pirlindole (pyrazinocarbazole) on the redox co-factor (flavin adenine dinucleotide, FAD) is a key factor in the inhibition. The kinetic, spectral, and thermodynamic changes induced by four closely related pirlindole analogues have been determined to investigate their interaction with the FAD in the active site of MAO A. For a model of flavin-inhibitor stacking, more favourable association would be expected between lumiflavin and the flatter analogues with a double bond at N3, and indeed lower K(i) values were found. However, the spectral changes induced by inhibitor binding to MAO A were 45% less for inhibitors with a double bond. Both in the absence and presence of the double bond, compounds with cyclohexyl at C8 induced 85% larger decrease in absorbance at 500nm than did those with a methyl substituent. In contrast, the K(i) values for the cyclohexyl compounds were lower, indicating greater affinity despite the lower perturbation of the flavin spectrum. All inhibitors stabilised the semiquinone of the FAD when MAO A was titrated with dithionite and prevented further reduction. These results indicate that the active site of MAO A is far more sensitive to structural variation than would be predicted by the simple flavin stacking model. Further, the independent changes in inhibitory potency and flavin perturbation preclude direct interaction with the flavin as a mode of binding and indicate that inhibitor-protein interactions must be important for inhibition.