Hans Wienk

New insights into the structure and composition of technical lignins: a comparative characterisation study

Detailed insight into the structure and composition of industrial (technical) lignins is needed to devise efficient thermal, bio- or chemocatalytic valorisation strategies. Six such technical lignins covering three main industrial pulping methods (Indulin AT Kraft, Protobind 1000 soda lignin and Alcell, poplar, spruce and wheat straw organosolv lignins) were comprehensively characterised by lignin composition analysis, FT-IR, pyrolysis-GC-MS, quantitative 31P and 2D HSQC NMR analysis and molar mass distribution by Size Exclusion Chromatography (SEC). A comparison of nine SEC methods, including the first analysis of lignins with commercial alkaline SEC columns, showed molar masses to vary considerably, allowing some recommendations to be made. The lignin molar mass decreased in the order: Indulin Kraft > soda P1000 > Alcell > OS-W ∼ OS-P ∼ OS-S, regardless of the SEC method chosen. Structural identification and quantification of aromatic units and inter-unit linkages indicated that all technical lignins, including the organosolv ones, have considerably been degraded and condensed by the pulping process. Importantly, low amounts of β- ether linkages were found compared to literature values for protolignin and lignins obtained by other, milder isolation processes. Stilbenes and ether furfural units could also be identified in some of the lignins. Taken together, the insights gained in the structure of the technical lignins, in particular, the low β-O-4 contents, carry implications for the design of lignin valorisation strategies including (catalytic) depolymerisation and material applications.

The structural flexibility of the preferredoxin transit peptide

In order to obtain insight into the structural flexibility of chloroplast targeting sequences, the Silene pratensis preferredoxin transit peptide was studied by circular dichroism and nuclear magnetic resonance spectroscopy. In water, the peptide is unstructured, with a minor propensity towards helix formation from Val‐9 to Ser‐12 and from Gly‐30 to Ser‐40. In 50% (v/v) trifluoroethanol, structurally independent N‐ and C‐terminal helices are stabilized. The N‐terminal helix appears to be amphipathic, with hydrophobic and hydroxylated amino acids on opposite sides. The C‐terminal helix comprises amino acids Met‐29–Gly‐50 and is destabilized at Gly‐39. No ordered tertiary structure was observed. The results are discussed in terms of protein import into chloroplasts, in which the possible interactions between the transit peptide and lipids are emphasized.

Structural studies of the exopolysaccharide produced by Lactobacillus paracasei 34-1.

The exopolysaccharide produced by Lactobacillus paracasei 34-1 in a semi-defined medium was found to be a heteropolymer, composed of D-galactose, 2-acetamido-2-deoxy-D-galactose, and sn-glycerol 3-phosphate in molar ratios of 3:1:1. By means of deglycerophosphorylation, methylation analysis, and 1D/2D NMR studies (1H, 13C, and 31P) the polysaccharide was shown to consist of repeating units with the following structure: [formula: see text].

Crystal structure and collagen-binding site of immune inhibitory receptor LAIR-1: unexpected implications for collagen binding by platelet receptor GPVI

Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1), one of the most widely spread immune receptors, attenuates immune cell activation when bound to specific sites in collagen. The collagen-binding domain of LAIR-1 is homologous to that of glycoprotein VI (GPVI), a collagen receptor crucial for platelet activation. Because LAIR-1 and GPVI also display overlapping collagen-binding specificities, a common structural basis for collagen recognition would appear likely. Therefore, it is crucial to gain insight into the molecular interaction of both receptors with their ligand to prevent unwanted cross-reactions during therapeutic intervention. We determined the crystal structure of LAIR-1 and mapped its collagen-binding site by nuclear magnetic resonance (NMR) titrations and mutagenesis. Our data identify R59, E61, and W109 as key residues for collagen interaction. These residues are strictly conserved in LAIR-1 and GPVI alike; however, they are located outside the previously proposed GPVI collagen-binding site. Our data provide evidence for an unanticipated mechanism of collagen recognition common to LAIR-1 and GPVI. This fundamental insight will contribute to the exploration of specific means of intervention in collagen-induced signaling in immunity and hemostasis.

E2–c-Cbl Recognition Is Necessary but not Sufficient for Ubiquitination Activity

The E2 ubiquitin-conjugating enzymes UbcH7 and UbcH5B both show specific binding to the RING (really interesting new gene) domain of the E3 ubiquitin-protein ligase c-Cbl, but UbcH7 hardly supports ubiquitination of c-Cbl and substrate in a reconstituted system. Here, we found that neither structural changes nor subtle differences in the E2-E3 interaction surface are possible explanations for the functional specificity of UbcH5B and UbcH7 in their interaction with c-Cbl. The quick transfer of ubiquitin from the UbcH5B-Ub thioester to c-Cbl or other ubiquitin acceptors suggests that UbcH5B might functionally be a relatively pliable E2 enzyme. In contrast, the UbcH7-Ub thioester is too stable to transfer ubiquitin under our assay conditions, indicating that UbcH7 might be a more specific E2 enzyme. Our results imply that the interaction specificity between c-Cbl and E2 is required but not sufficient for transfer of ubiquitin to potential targets.

Structural Dynamics in the Activation of Epac

Epac1 is a cAMP-responsive exchange factor for the small G-protein Rap. It consists of a regulatory region containing a cyclic nucleotide binding (CNB) domain and a catalytic region that activates Rap. In the absence of cAMP, access of Rap to the catalytic site is blocked by the regulatory region. We analyzed the conformational states of the CNB domain in the absence and in the presence of cAMP and cAMP analogues by NMR spectroscopy, resulting in the first direct insights into the activation mechanism of Epac. We prove that the CNB domain exists in equilibrium between the inactive and the active conformation, which is shifted by binding of cAMP. cAMP binding results in conformational changes in both the ligand binding pocket and the outer helical segments. We used two different cAMP antagonists that block these successive changes to elucidate the steps of this process. Highlighting the role of dynamics, the superactivator 8-pCPT-2′-O-Me-cAMP induces similar conformational changes as cAMP but causes different internal mobility. The results reveal the critical elements of the CNB domain of Epac required for activation and highlight the role of dynamics in this process.

Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: Factors contributing to increased activity retention

Previously, Lipase A from Bacillus subtilis was subjected to in vitro directed evolution using iterative saturation mutagenesis, with randomization sites chosen on the basis of the highest B‐factors available from the crystal structure of the wild‐type (WT) enzyme. This provided mutants that, unlike WT enzyme, retained a large part of their activity after heating above 65°C and cooling down. Here, we subjected the three best mutants along with the WT enzyme to biophysical and biochemical characterization. Combining thermal inactivation profiles, circular dichroism, X‐ray structure analyses and NMR experiments revealed that mutations of surface amino acid residues counteract the tendency of Lipase A to undergo precipitation under thermal stress. Reduced precipitation of the unfolding intermediates rather than increased conformational stability of the evolved mutants seems to be responsible for the activity retention.

Specificity and affinity of Lac repressor for the auxiliary operators O2 and O3 are explained by the structures of their protein-DNA complexes.

The structures of a dimeric mutant of the Lac repressor DNA-binding domain complexed with the auxiliary operators O2 and O3 have been determined using NMR spectroscopy and compared to the structures of the previously determined Lac-O1 and Lac-nonoperator complexes. Structural analysis of the Lac-O1 and Lac-O2 complexes shows highly similar structures with very similar numbers of specific and nonspecific contacts, in agreement with similar affinities for these two operators. The left monomer of the Lac repressor in the Lac-O3 complex retains most of these specific contacts. However, in the right half-site of the O3 operator, there is a significant loss of protein-DNA contacts, explaining the low affinity of the Lac repressor for the O3 operator. The binding mode in the right half-site resembles that of the nonspecific complex. In contrast to the Lac-nonoperator DNA complex where no hinge helices are formed, the stability of the hinge helices in the weak Lac-O3 complex is the same as in the Lac-O1 and Lac-O2 complexes, as judged from the results of hydrogen/deuterium experiments.

Solution structure of the C1-subdomain of Bacillus stearothermophilus translation initiation factor IF2.

IF2 is one of three bacterial translation initiation factors that are conserved through all kingdoms of life. It binds the 30S and 50S ribosomal subunits, as well as fMet‐tRNAfMet. After these interactions, fMet‐tRNAfMet is oriented to the ribosomal P‐site where the first amino acid of the nascent polypeptide, formylmethionine, is presented. The C‐terminal domain of Bacillus stearothermophilus IF2, which is responsible for recognition and binding of fMet‐tRNAfMet, contains two structured modules. Previously, the solution structure of the most C‐terminal module, IF2‐C2, has been elucidated by NMR spectroscopy and direct interactions between this subdomain and fMet‐tRNAfMet were reported. In the present NMR study we have obtained the spectral assignment of the other module of the C‐terminal domain (IF2‐C1) and determined its solution structure and backbone dynamics. The IF2‐C1 core forms a flattened fold consisting of a central four‐stranded parallel β‐sheet flanked by three α‐helices. Although its overall organization resembles that of subdomain III of the archaeal IF2‐homolog eIF5B whose crystal structure had previously been reported, some differences of potential functional significance are evident.

Structure-Guided Design of Selective Epac1 and Epac2 Agonists

The second messenger cAMP is known to augment glucose-induced insulin secretion. However, its downstream targets in pancreatic β-cells have not been unequivocally determined. Therefore, we designed cAMP analogues by a structure-guided approach that act as Epac2-selective agonists both in vitro and in vivo. These analogues activate Epac2 about two orders of magnitude more potently than cAMP. The high potency arises from increased affinity as well as increased maximal activation. Crystallographic studies demonstrate that this is due to unique interactions. At least one of the Epac2-specific agonists, Sp-8-BnT-cAMPS (S-220), enhances glucose-induced insulin secretion in human pancreatic cells. Selective targeting of Epac2 is thus proven possible and may be an option in diabetes treatment.