Biology subjects

Garratt, R. C.

Publications and source records attributed to Garratt, R. C..

6 recordsLinked to original sources

bioRxiv2026

Coiled-coil homo-oligomerization and disaggregase Hsp104 act in parallel to stabilize orphan septins

Multiple septin family proteins co-assemble with strict subunit stoichiometry into hetero-oligomers. In the absence of native septin partners, purified septins aggregate in vitro, and "orphan" septins are found in pathological aggregates associated with neurodegenerative diseases. Cytosolic chaperones bind the septin GTPase domain to promote on-pathway septin folding but it was unclear how cells manage orphan septins to maintain septin subunit stoichiometry. Most septins have C-terminal domains (CTDs) that form heteromeric coiled coils within or between septin complexes. Here we present evidence that orphan yeast septins are protected from proteasomal degradation by forming transient coiled-coil homodimers and trimers and, in parallel, by the disaggregase chaperone Hsp104. Septins unable to undergo CTD-mediated homo-oligomerization require Hsp104 to accumulate to super-stoichiometric levels. We show that the number of septin-encoding mRNAs per yeast cell is low and variable, creating opportunities for transient subunit imbalances. These findings reveal a novel role for coiled coils and the cellular proteostasis machinery in the fidelity of higher-order septin assembly.

molecular biology↗

bioRxiv2023

A Key Piece of the Puzzle: The central tetramer of the Saccharomyces cerevisiae septin protofilament and Its Implications for Self-Assembly

Septins, often described as the fourth component of the cytoskeleton, are structural proteins found in a vast variety of living beings. They are related to small GTPases and thus, generally, present GTPase activity which may play an important (although incompletely understood) role in their organization and function. Septins polymerase into long non-polar filaments, in which each subunit interacts with two others by alternating interfaces, NC and G. In Saccharomyces cerevisiae four septins are organized in the following manner, [Cdc11-Cdc12-Cdc3-Cdc10- Cdc10-Cdc3-Cdc12-Cdc11]n in order to form filaments. Although septins were originally discovered in yeast and much is known regarding their biochemistry and function, only limited structural information about them is currently available. Here we present crystal structures of Cdc3/Cdc10 which provide the first view of the physiological interfaces formed by yeast septins. The G-interface has properties which place it in between that formed by SEPT2/SEPT6 and SEPT7/SEPT3 in human filaments. Switch I from Cdc10 contributes significantly to the interface, whereas in Cdc3 it is largely disorded. However, the significant negative charge density of the latter suggests it may have a unique role. At the NC-interface, we describe an elegant means by which the sidechain of a glutamine from helix 0 imitates a peptide group in order to retain hydrogen-bond continuity at the kink between helices 5 and 6 in the neighbouring subunit, thereby justifying the conservation of the helical distortion. Its absence from Cdc11, along with this structures other unusual features are critically discussed by comparison with Cdc3 and Cdc10. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/537027v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@10c1eccorg.highwire.dtl.DTLVardef@ba0dacorg.highwire.dtl.DTLVardef@d7b866org.highwire.dtl.DTLVardef@8b6d7e_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIThe first crystal structure of a yeast septin heterodimer (Cdc3-Cdc10) provides important insights into their structural biology. C_LIO_LIIdentification of common features and differences between yeast and human septins, sheds light on the unique characteristics of yeast septin filaments. C_LIO_LIThe Cdc3G-Cdc10{Delta}1-10 crystal structure could be a crucial piece of the puzzle towards obtaining a high-resolution cryo-EM structure of the yeast septin octamer. C_LI

molecular biology↗

bioRxiv2022

Structural Characterization of L-Galactose Dehydrogenase: A Key Enzyme for Vitamin C Biosynthesis

In plants, it is well-known that ascorbic acid (vitamin C) can be synthesized via multiple metabolic pathways but there is still much to be learnt concerning their integration and control mechanisms. Furthermore, the structural biology of the component enzymes has been poorly exploited. Here we describe the first crystal structure for an L-galactose dehydrogenase (SoGDH from spinach), from the D-mannose/L-galactose (Smirnoff Wheeler) pathway which converts L-galactose into L-galactono-1,4-lactone. The kinetic parameters for the enzyme are similar to those from its homologue from camu-camu, a super-accumulator of vitamin C found in the Peruvian amazon. Both enzymes are monomers in solution, have a pH optimum of 7 and their activity is largely unaffected by high concentrations of ascorbic acid, suggesting the absence of a feedback mechanism acting via GDH. Previous reports may have been influenced by changes of the pH of the reaction medium as a function of ascorbic acid concentration. The structure of SoGDH is dominated by a ({beta}/)8 barrel closely related to aldehyde-keto reductases (AKRs). The structure bound to NAD+ shows that the lack of Arg279 justifies its preference for NAD+ over NADP+, as employed by many AKRs. This favours the oxidation reaction which ultimately leads to ascorbic acid accumulation. When compared with other AKRs, residue substitutions at the C-terminal end of the barrel (Tyr185, Tyr61, Ser59 and Asp128) can be identified to be likely determinants of substrate specificity. The present work contributes towards a more comprehensive understanding of structure-function relationships in the enzymes involved in vitamin C synthesis.

biophysics↗

bioRxiv2020

Structural and functional analysis of pyocin S8 from Pseudomonas aeruginosa : requirement of a glutamate in the H-N-H motif for the DNase activity.

Multi-drug resistance (MDR) is a serious threat to global public health, making the development of new antimicrobials an urgent necessity. Pyocins are protein antibiotics produced by Pseudomonas aeruginosa strains to kill closely related cells during intraspecific competition. Here, we report an in depth biochemical, microbicidal and structural characterization of a new S-type pyocin, named S8. Initially, we described the domain organization and secondary structure of S8. Subsequently, we observed that a recombinant S8 composed of the killing subunit in complex with the immunity (Im) protein killed the strain PAO1. Furthermore, mutation of a highly conserved glutamic acid to alanine (Glu100Ala) completely inhibited this antimicrobial activity. Probably the integrity of the H-N-H motif is essential in the killing activity of S8, as Glu100 is a highly conserved component of this structure. Next, we observed that S8 is a metal-dependent endonuclease, as EDTA treatment abolished its ability to cleave supercoiled pUC18 plasmid. Supplementation of apo S8 with Ni2+ strongly induced this DNase activity, whereas Mn2+ and Mg2+ exhibited moderate effects and Zn2+ was inhibitory. Additionally, S8 bound Zn2+ with a higher affinity than Ni2+ and the Glu100Ala mutation decreased the affinity of S8 for these metals as shown by isothermal titration calorimetry (ITC). Finally, we describe the crystal structure of the Glu100Ala pyocin-S8DNase-Im complex at 1.38 [A], which gave us new insights into the endonuclease activity of S8. Our results reinforce the possible use of S8 as an alternative antibiotic for MDR Pseudomonas aeruginosa strains, while leaving commensal human microbiota intact.

molecular biology↗

bioRxiv2019

Repositioning septins within the core particle

Septins are GTP binding proteins considered to be a novel component of the cytoskeleton. They polymerize into filaments based on hetero-oligomeric core particles which, in humans, are either hexamers or octamers composed of two copies each of either three or four different septins from the 13 available. Not all combinations are possible as it is believed that these must obey substitution rules which determine that different septins must be derived from four distinct and well-established sub-groups. Here, we have purified and characterized one such combinations, SEPT5-SEPT6-SEPT7, and used TEM to derive the first structural information concerning its assembly. The full complex was purified using an affinity tag attached to only one of its components (SEPT7) and was able to bind to and perturb lipid bilayers. Although the complex assembled into elongated hexameric particles, the position of SEPT5 was incompatible with that predicted by the reported structure of SEPT2-SEPT6-SEPT7 based on the substitution rules. MBP-fusion constructs for SEPT5 and SEPT2 and immuno-staining clearly show that these septins occupy the terminal positions of the SEPT5-SEPT6-SEPT7 and SEPT2-SEPT6-SEPT7 hexamers, respectively. In so doing they expose a so-called NC interface which we show to be more susceptible to perturbation at high salt concentrations. Our results show that the true structure of the hexamer is inverted with respect to that described previously and, as such, is more compatible with that reported for yeast. Taken together, our results suggest that the mechanisms involved in spontaneous self-assembly of septin core particles and their filaments deserve further reflection.

biochemistry↗

bioRxiv2019

Septin 9-containging filaments and Golgi assembly depend on two polybasic domains

Septins are GTP-binding proteins involved in several membrane remodeling mechanisms. They associate with membranes, presumably by using a polybasic domain (PB1) that interacts with phosphoinositides (PIs). Membrane-bound septins assemble into microscopic structures that regulate membrane shape. How septins exactly interact with PIs, assemble, and shape membranes is weakly understood. Here, we found that septin 9 has a second polybasic domain (PB2) conserved in the human septin family. Similarly to PB1, PB2 binds specifically to PIs, and both domains are critical for septin filament formation. However, septin 9 membrane association does not depend on these PB domains but on putative PB-adjacent amphipathic helices. The presence of the PB domains guarantees the protein enrichment to PI-contained membranes, which is critical for PI-enriched organelles. In particular, we found that septin 9 PB domains control the assembly and functionality of the Golgi apparatus. Our findings bring novel insights into the role of septins in organelle morphology. HighlightsO_LITwo polybasic domains mediate septin 9 interaction with PIs C_LIO_LIHuman septins have amphipathic helices suitable for binding membrane C_LIO_LISeptin 9 polybasic domains mediate septin high order structure formation C_LIO_LIMutation or depletion of septin polybasic domains induce Golgi fragmentation C_LI

cell biology↗