Biology subjects

Ubbink, M.

Publications and source records attributed to Ubbink, M..

6 recordsLinked to original sources

bioRxiv2021

Novel anti-repression mechanism of H-NS proteins by a phage "early protein".

H-NS family proteins, bacterial xenogeneic silencers, play central roles in genome organization and in the regulation of foreign genes. It is thought that gene repression is directly dependent on the DNA binding modes of H-NS family proteins. These proteins form lateral protofilaments along DNA. Under specific environmental conditions they switch to bridging two DNA duplexes. This switching is a direct effect of environmental conditions on electrostatic interactions between the oppositely charged DNA binding and N-terminal domains of H-NS proteins. The Pseudomonas lytic phage LUZ24 encodes the early protein gp4, which modulates the DNA binding and function of the H-NS family protein MvaT of Pseudomonas aeruginosa. However, the mechanism by which gp4 modulates MvaT activity remains elusive. In this study, we show that gp4 specifically interferes with the formation and stability of the bridged MvaT-DNA complex. Structural investigations suggest that gp4 acts as an "electrostatic zipper" between the oppositely charged domains of MvaT protomers, and stabilizes a structure resembling their "half-open" conformation, resulting in relief of gene silencing and adverse effects on P. aeruginosa growth. The ability to control H-NS conformation and thereby its impact on global gene regulation and growth might open new avenues to fight Pseudomonas multidrug resistance.

biochemistry↗

bioRxiv2020

Tuning the transglycosylation reaction of a GH11 xylanase by a delicate enhancement of its thumb flexibility

Glycoside hydrolases (GH) are attractive tools for multiple biotechnological applications. In conjunction with their hydrolytic function, GH can perform transglycosylation reaction under specific conditions. In nature, oligosaccharides synthesis is performed by glycosyltransferase (GT). However, the industrial utilization of GT is limited by their instability in solution. A key difference between GT and GH is the flexibility of their binding sites architecture. In this report, we used the xylanase from Bacillus circulans (BCX) to study the interplay between active site flexibility and the transglycosylation reaction. Residues of the BCX thumb were substituted to increase the flexibility of the enzyme binding site. Replacement of the highly conserved residue P116 with glycine shifted the balance of the BCX enzymatic reaction toward transglycosylation. The effects of this point mutation on the structure and dynamics of BCX were investigated by NMR spectroscopy. The P116G mutation induces subtle changes in the configuration of the thumb and enhances the millisecond dynamics of the active site. Based on our findings, we propose the remodeling of the GH enzymes glycon site flexibility as a strategy to improve the transglycosylation efficiency of these biotechnologically important catalysts.

biochemistry↗

bioRxiv2020

The MPS1 kinase NTE region has helical propensity and preferred conformations towards the TPR domain

The mitotic spindle assembly checkpoint (SAC) ensures accurate segregation of chromosomes by preventing onset of anaphase until all chromosomes are properly attached to spindle microtubules. The Monopolar spindle 1 (MPS1) kinase is one of the SAC components, localizing at unattached kinetochores by an N-terminal localization module. This module comprises a flexible NTE module and the TPR domain, which we previously characterized for their contribution to kinetochore binding. Here we discuss the conformations of the highly flexible NTE with respect to the TPR domain, using paramagnetic NMR. The distance restraints derived from paramagnetic relaxation enhancements (PREs) show that the mobile NTE can be found in proximity of a large but specific part of the surface area of the TPR domain. To sample the conformational space of the NTE in the context of the NTE-TPR module, we used the ab initio Rosetta approach supplemented by paramagnetic NMR restraints. We find that many NTE residues have a propensity to form helical structures and that the module localizes at the convex surface of the TPR domain. This work demonstrates the highly dynamic nature of the interactions between the NTE and TPR domains and it shows that the convex rather than the canonical concave TPR surface mediates interactions, leading to the auto-inhibition that the TPR exerts upon the NTE region in the context of SAC signaling.

biochemistry↗

bioRxiv2019

Catalysis by a rigid enzyme

Many enzymes are dynamic entities, sampling conformational states that are relevant for catalytic activity. Crystal structures of catalytic intermediates suggest, however, that not all enzymes require structural changes for activity. The single-domain enzyme xylanase from Bacillus circulans (BCX) is involved in the degradation of hemicellulose. We demonstrate that BCX in solution undergoes minimal structural changes during catalysis. NMR spectroscopy results show that the rigid protein matrix provides a frame for fast substrate binding in multiple conformations, accompanied by slow, enzyme induced substrate distortion. Therefore, we propose a model in which the rigid enzyme takes advantage of substrate flexibility to induce a conformation that facilitates catalysis.\n\nOne Sentence SummaryThe rigid matrix of BCX uses substrate flexibility in Michaelis complex formation.

biochemistry↗

bioRxiv2019

Structural basis for osmotic regulation of the DNA binding properties of H-NS proteins

H-NS proteins act as osmotic sensors translating changes in osmolarity into altered DNA binding properties, thus, regulating enterobacterial genome organization and genes transcription. The molecular mechanism underlying the switching process and its conservation among H-NS family members remains elusive.\n\nHere, we focus on the H-NS family protein MvaT from P. aeruginosa and demonstrate experimentally that its protomer exists in two different conformations, corresponding to two different functional states. In the half-opened state (dominant at low salt) the protein forms filaments along DNA, in the fully opened state (dominant at high salt) the protein bridges DNA. This switching is a direct effect of ionic strengths on electrostatic interactions between the appositively charged DNA binding and N-terminal domains of MvaT. The asymmetric charge distribution and intramolecular interactions are conserved among the H-NS family of proteins. Therefore, our study establishes a general paradigm for the molecular mechanistic basis of the osmosensitivity of H-NS proteins.

biochemistry↗

bioRxiv2016

Structural and Functional Characterization of the Alanine Racemase from Streptomyces coelicolor A3(2)

The conversion of L-alanine (L-Ala) into D-alanine (D-Ala) in bacteria is performed by pyridoxal phosphate-dependent enzymes called alanine racemases. D-Ala is an essential component of the bacterial peptidoglycan and hence required for survival. The Gram-positive bacterium Streptomyces coelicolor has at least one alanine racemase encoded by alr. Here, we describe an alr deletion mutant of S. coelicolor which depends on D-Ala for growth and shows increased sensitivity to the antibiotic D-cycloserine (DCS). The crystal structure of the alanine racemase (Alr) was solved with and without the inhibitors DCS or propionate, at 1.64 [A] and 1.51 [A] resolution, respectively. The crystal structures revealed that Alr is a homodimer with residues from both monomers contributing to the active site. The dimeric state of the enzyme in solution was confirmed by gel filtration chromatography, with and without L-Ala or D-cycloserine. Specificity of the enzyme was 66 +/- 3 U mg-1 for the racemization of L-to D-Ala, and 104 +/- 7 U mg-1 for the opposite direction. Comparison of Alr from S. coelicolor with orthologous enzymes from other bacteria, including the closely related D-cycloserine-resistant Alr from S. lavendulae, strongly suggests that structural features such as the hinge angle or the surface area between the monomers do not contribute to D-cycloserine resistance, and the molecular basis for resistance therefore remains elusive.

molecular biology↗