A dynamically interacting flexible loop assists oligomerisation of the Caenorhabditis elegans centriolar protein SAS-6.

Centrioles are conserved organelles fundamental for the organisation of microtubules in animal cells. Oligomerisation of the spindle assembly abnormal protein 6 (SAS-6) is an essential step in the centriole assembly process and may act as trigger for the formation of these organelles. SAS-6 oligomerisation is driven by two independent interfaces, comprising an extended coiled coil and a dimeric N-terminal globular domain. However, how SAS-6 oligomerisation is controlled remains unclear. Here, we show that in the Caenorhabditis elegans SAS-6, a segment of the N-terminal globular domain, unresolved in crystallographic structures, comprises a flexible loop that assists SAS-6 oligomerisation. Atomistic molecular dynamics simulations and nuclear magnetic resonance experiments suggest that transient interactions of this loop across the N-terminal dimerisation interface stabilise the SAS-6 oligomer. We discuss the possibilities presented by such flexible SAS-6 segments for the control of centriole formation.

Base-Independent DNA Base-Excision Repair of 8-Oxoguanine.

Living organisms protect their genome from gene mutation by excising damaged DNA bases. Here, 8-oxoguanine (8OG) is one of the most abundant DNA lesions. In bacteria the base excision is catalyzed by the enzyme formamidopyrimidine-DNA- glycosylase (Fpg), for which two different orientations of 8OG binding into the active site of Fpg have been proposed: syn- and anti-conformation. Here, we present a new ribose-protonated repair mechanism for 8OG that is base-independent and can excise 8OG in both conformations. Using high-level QM/MM calculations with up to 588/573 atoms in the QM sphere, the activation barrier is computed in excellent agreement with the experimentally measured value. Since the excised base itself is not directly involved in the mechanism, this implies that lesion discrimination does not occur within the active site of the enzyme.

Structural, Biochemical, and Computational Studies Reveal the Mechanism of Selective Aldehyde Dehydrogenase 1A1 Inhibition by Cytotoxic Duocarmycin Analogues.

Analogues of the natural product duocarmycin bearing an indole moiety were shown to bind aldehyde dehydrogenase 1A1 (ALDH1A1) in addition to DNA, while derivatives without the indole solely addressed the ALDH1A1 protein. The molecular mechanism of selective ALDH1A1 inhibition by duocarmycin analogues was unraveled through cocrystallization, mutational studies, and molecular dynamics simulations. The structure of the complex shows the compound embedded in a hydrophobic pocket, where it is stabilized by several crucial π-stacking and van der Waals interactions. This binding mode positions the cyclopropyl electrophile for nucleophilic attack by the noncatalytic residue Cys302, thereby resulting in covalent attachment, steric occlusion of the active site, and inhibition of catalysis. The selectivity of duocarmycin analogues for ALDH1A1 is unique, since only minor alterations in the sequence of closely related protein isoforms restrict compound accessibility.

A base-independent repair mechanism for DNA glycosylase--no discrimination within the active site.

The ubiquitous occurrence of DNA damages renders its repair machinery a crucial requirement for the genomic stability and the survival of living organisms. Deficiencies in DNA repair can lead to carcinogenesis, Alzheimer, or Diabetes II, where increased amounts of oxidized DNA bases have been found in patients. Despite the highest mutation frequency among oxidized DNA bases, the base-excision repair process of oxidized and ring-opened guanine, FapydG (2,6-diamino-4-hydroxy-5-formamidopyrimidine), remained unclear since it is difficult to study experimentally. We use newly-developed linear-scaling quantum-chemical methods (QM) allowing us to include up to 700 QM-atoms and achieving size convergence. Instead of the widely assumed base-protonated pathway we find a ribose-protonated repair mechanism which explains experimental observations and shows strong evidence for a base-independent repair process. Our results also imply that discrimination must occur during recognition, prior to the binding within the active site.

Kinetic and Theoretical Studies of Beta-Lactone Reactivity-A Quantitative Scale for Biological Application.

Natural products comprise a rich source for bioactive molecules with medicinal relevance. Many of these contain electrophilic scaffolds that bind conserved enzyme active sites covalently. Prominent examples include beta-lactams and beta-lactones, which specifically acylate serine residues in diverse peptidases. Although these scaffolds appear similar, their bioactivities and corresponding protein targets vary. To quantify and dissect these differences in bioactivities, the kinetics of the reactions of beta-butyrolactone with a set of reference amines in buffered aqueous solution at 37 °C have been analyzed. Different product ratios of C1 versus C3 attack on the beta-butyrolactone have been observed, depending on the aliphatic or aromatic nature of the standard amine used. Quantum mechanics/molecular mechanics (QM/MM) calculations reveal that a H3 O+ molecule has a crucial role in stabilizing C3 attack by aniline, through coordination of the lactone ring oxygen. In agreement with their weak proteome reactivity, monocyclic beta-lactams did not react with the set of standard nucleophiles studied herein. Bicyclic beta-lactams, however, exhibited a lower activation barrier, and thus, reacted with standard nucleophiles. This study represents a starting point for semiquantitative reactivity scales for natural products, which, in analogy to chemical reactivity scales, will provide predictions for electrophilic modifications in biological systems.

Ribose-protonated DNA base excision repair: a combined theoretical and experimental study.

Living organisms protect the genome against external influences by recognizing and repairing damaged DNA. A common source of gene mutation is the oxidized guanine, which undergoes base excision repair through cleavage of the glycosidic bond between the ribose and the nucleobase of the lesion. We unravel the repair mechanism utilized by bacterial glycosylase, MutM, using quantum-chemical calculations involving more than 1000 atoms of the catalytic site. In contrast to the base-protonated pathway currently favored in the literature, we show that the initial protonation of the lesion's ribose paves the way for an almost barrier-free glycosidic cleavage. The combination of theoretical and experimental data provides further insight into the selectivity and discrimination of MutM's binding site toward various substrates.

Caenorhabditis elegans centriolar protein SAS-6 forms a spiral that is consistent with imparting a ninefold symmetry.

Centrioles are evolutionary conserved organelles that give rise to cilia and flagella as well as centrosomes. Centrioles display a characteristic ninefold symmetry imposed by the spindle assembly abnormal protein 6 (SAS-6) family. SAS-6 from Chlamydomonas reinhardtii and Danio rerio was shown to form ninefold symmetric, ring-shaped oligomers in vitro that were similar to the cartwheels observed in vivo during early steps of centriole assembly in most species. Here, we report crystallographic and EM analyses showing that, instead, Caenorhabotis elegans SAS-6 self-assembles into a spiral arrangement. Remarkably, we find that this spiral arrangement is also consistent with ninefold symmetry, suggesting that two distinct SAS-6 oligomerization architectures can direct the same output symmetry. Sequence analysis suggests that SAS-6 spirals are restricted to specific nematodes. This oligomeric arrangement may provide a structural basis for the presence of a central tube instead of a cartwheel during centriole assembly in these species.