Biology subjects

Leonardo, D. A.

Publications and source records attributed to Leonardo, D. A..

3 recordsLinked to original sources

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↗

bioRxiv2021

Identification of unknown proteins in X-ray crystallography and cryo-EM

Although experimental protein structure determination usually targets known proteins, chains of unknown sequence are often encountered. They can be purified from natural sources, appear as an unexpected fragment of a well characterized protein or as a contaminant. Regardless of the source of the problem, the unknown protein always requires tedious characterization. Here we present an automated pipeline for the identification of protein sequences from cryo-EM reconstructions and crystallographic data. We present the methods application to characterize the crystal structure of an unknown protein purified from a snake venom. We also show that the approach can be successfully applied to the identification of protein sequences and validation of sequence assignments in cryo-EM protein structures.

molecular biology↗