Glutamate 270 plays an essential role in K activation and domain closure of Thermus thermophilus isopropylmalate dehydrogenase

The mutant E270A of Thermus thermophilus 3-isopropylmalate dehydrogenase exhibits largely reduced (∼1%) catalytic activity and negligible activation by K+ compared to the wild-type enzyme. A 3–4 kcal/mol increase in the activation energy of the catalysed reaction upon this mutation could also be predicted by QM/MM calculations. In the X-ray structure of the E270A mutant a water molecule was observed to take the place of K+. SAXS and FRET experiments revealed the essential role of E270 in stabilisation of the active domain-closed conformation of the enzyme. In addition, E270 seems to position K+ into close proximity of the nicotinamide ring of NAD+ and the electron-withdrawing effect of K+ may help to polarise the aromatic ring in order to aid the hydride-transfer

A small angle x-ray scattering study of the droplet-cylinder transition in oil-rich sodium bis(2-ethylhexyl) sulfosuccinate microemulsions

Supramolecular & Biomaterials Chemistr

Rigidity, conformation and solvation of native and oxidized tannin macromolecules in water-ethanol solution

International audienc

Interactions of ataxin-3 with its molecular partners in the protein machinery that sorts protein aggregates to the aggresome

Ataxin-3 (AT3) is the protein that triggers the inherited neurodegenerative disorder spinocerebellar ataxia type 3 when its polyglutamine (polyQ) stretch close to the C-terminus exceeds a critical length. AT3 consists of the N-terminal globular Josephin domain (JD) and the C-terminal disordered one. It cleaves isopeptide bonds between ubiquitin monomers, an event involved in protein quality control mechanisms. AT3 has been implicated in the pathway that sorts aggregated protein to aggresomes via microtubules, in which dynein and histone deacetylase 6 (HDAC6) also seem to be involved. By taking advantage of small angle X-ray scattering (SAXS) and surface plasmon resonance (SPR), we have investigated the interaction of AT3 with tubulin and HDAC6. Based on SAXS results, the AT3 oligomer, consisting of 6-7 subunits, tightly binds to the tubulin hexameric oligomer in a "parallel" fashion. By SPR analysis we have demonstrated that AT3 binds to tubulin dimer with a 50 nM affinity. Binding fits with a Langmuir 1:1 model and involves a single binding interface. Nevertheless, the interaction surface consists of three distinct, discontinuous tubulin-binding regions (TBR), one located in the JD, and the two others in the disordered domain, upstream and downstream of the polyQ stretch. In the absence of any of the three TBRs, the affinity is drastically reduced. By SPR we have also provided the first evidence of direct binding of AT3 to HDAC6, with affinity in the range 0.1-1 \u3bcM. These results shed light on the interactions among the components of the transport machinery that sorts aggregate protein to the aggresome, and pave the way to in vivo studies aimed at further clarifying their roles

3D-printed microfluidic system for the in situ diagnostics and screening of nanoparticles synthesis parameters

Fine tuning of the material properties requires many trials and errors during the synthesis. The metal nanoparticles undergo several stages of reduction, clustering, coalescence and growth upon their formation. Resulting properties of the colloidal solution thus depend on the concentrations of the reagents, external temperature, synthesis protocol and qualification of the researcher determines the reproducibility and quality. Automatized flow systems overcome the difficulties inherent for the conventional batch approaches. Microfluidic systems represent a good alternative for the high throughput data collection. The recent advances in 3D-printing made complex topologies in microfluidic devices cheaper and easily customizable. However, channels of the cured photopolymer resin attract metal ions upon synthesis and create crystallization centers. In our work we present 3D-printed system for the noble metal nanoparticle synthesis in slugs. Alternating flows of oil and aqueous reaction mixtures prevent metal deposition on the channel walls. Elongated droplets are convenient for optical and X-ray diagnostics using conventional methods. We demonstrate the work of the system using Ag nanoparticles synthesis for machine-learning assisted tuning of the plasmon resonance frequency

Dual Role of the Active Site Residues of Thermus thermophilus 3 Isopropylmalate Dehydrogenase Chemical Catalysis and Domain Closure

The key active site residues K185, Y139, D217, D241, D245, and N102 of Thermus thermophilus 3 isopropylmalate dehydrogenase Tt IPMDH have been replaced, one by one, with Ala. A drastic decrease in the kcat value 0.06 compared to that of the wild type enzyme has been observed for the K185A and D241A mutants. Similarly, the catalytic interactions Km values of these two mutants with the substrate IPM are weakened by more than 1 order of magnitude. The other mutants retained some 1 amp; 8722;13 of the catalytic activity of the wild type enzyme and do not exhibit appreciable changes in the substrate Km values. The pH dependence of the wild type enzyme activity pK 7.4 is shifted toward higher values for mutants K185A and D241A pK values of 8.4 and 8.5, respectively . For the other mutants, smaller changes have been observed. Consequently, K185 and D241 may constitute a proton relay system that can assist in the abstraction of a proton from the OH group of IPM during catalysis. Molecular dynamics simulations provide strong support for the neutral character of K185 in the resting state of the enzyme, which implies that K185 abstracts the proton from the substrate and D241 assists the process via electrostatic interactions with K185. Quantum mechanics molecular mechanics calculations revealed a significant increase in the activation energy of the hydride transfer of the redox step for both D217A and D241A mutants. Crystal structure analysis of the molecular contacts of the investigated residues in the enzyme amp; 8722;substrate complex revealed their additional importance in particular that of K185, D217, and D241 in stabilizing the domain closed active conformation. In accordance with this, small angle X ray scattering measurements indicated the complete absence of domain closure in the cases of D217A and D241A mutants, while only partial domain closure could be detected for the other mutants. This suggests that the same residues that are important for catalysis are also essential for inducing domain closur

The protealysin operon encodes emfourin, a prototype of a novel family of protein metalloprotease inhibitors

Protealysin is a Serratia proteamaculans metalloproteinase of the M4 peptidase family and the prototype of a large group of protealysin-like proteases (PLPs). PLPs are likely involved in bacterial interaction with plants and animals as well as in bacterial pathogenesis. We demonstrated that the PLP genes in bacteria colocalize with the genes of putative conserved proteins. In S. proteamaculans, these two genes form a bicistronic operon. The putative S. proteamaculans protein that we called emfourin (M4in) was expressed in Escherichia coli and characterized. M4in forms a complex with protealysin with a 1:1 stoichiometry and is a potent slow-binding competitive inhibitor of protealysin (Ki = 52 ± 14 pM); besides, M4in is not secreted from S. proteamaculans constitutively. A comparison of amino acid sequences of M4in and its homologs with those of known inhibitors suggests that M4in is the prototype of a new family of protein inhibitors of proteases

Identification of an N-terminal inhibitory extension as the primary mechanosensory regulator of twitchin kinase

Item does not contain fulltextTitin-like kinases are an important class of cytoskeletal kinases that intervene in the response of muscle to mechanical stimulation, being central to myofibril homeostasis and development. These kinases exist in autoinhibited states and, allegedly, become activated during muscle activity by the elastic unfolding of a C-terminal regulatory segment (CRD). However, this mechano-activation model remains controversial. Here we explore the structural, catalytic, and tensile properties of the multidomain kinase region of Caenorhabditis elegans twitchin (Fn(31)-Nlinker-kinase-CRD-Ig(26)) using X-ray crystallography, small angle X-ray scattering, molecular dynamics simulations, and catalytic assays. This work uncovers the existence of an inhibitory segment that flanks the kinase N-terminally (N-linker) and that acts synergistically with the canonical CRD tail to silence catalysis. The N-linker region has high mechanical lability and acts as the primary stretch-sensor in twitchin kinase, while the CRD is poorly responsive to pulling forces. This poor response suggests that the CRD is not a generic mechanosensor in this kinase family. Instead, the CRD is shown here to be permissive to catalysis and might protect the kinase active site against mechanical damage. Thus, we put forward a regulatory model where kinase inhibition results from the combined action of both N- and C-terminal tails, but only the N-terminal extension undergoes mechanical removal, thereby affording partial activation. Further, we compare invertebrate and vertebrate titin-like kinases and identify variations in the regulatory segments that suggest a mechanical speciation of these kinase classes