Crystal structure of CTP:glycerol-3-phosphate cytidylyltransferase from Staphylococcus aureus: Examination of structural basis for kinetic mechanism

AbstractTransporters play vital roles in acquiring antimicrobial resistance among pathogenic bacteria. In this study, we report the X-ray structure of NorC, a 14-transmembrane major facilitator superfamily member that is implicated in fluoroquinolone resistance in drug-resistant Staphylococcus aureus strains, at a resolution of 3.6 Å. The NorC structure was determined in complex with a single-domain camelid antibody that interacts at the extracellular face of the transporter and stabilizes it in an outward-open conformation. The complementarity determining regions of the antibody enter and block solvent access to the interior of the vestibule, thereby inhibiting alternating-access. NorC specifically interacts with an organic cation, tetraphenylphosphonium, although it does not demonstrate an ability to transport it. The interaction is compromised in the presence of NorC-antibody complex, consequently establishing a strategy to detect and block NorC and related transporters through the use of single-domain camelid antibodies.

Download Full-text

Structural basis of Naa20 activity towards a canonical NatB substrate

Communications Biology ◽

10.1038/s42003-020-01546-4 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Dominik Layer ◽

Jürgen Kopp ◽

Miriam Fontanillo ◽

Maja Köhn ◽

Karine Lapouge ◽

...

Keyword(s):

Crystal Structure ◽

Drug Development ◽

Catalytic Mechanism ◽

Peptide Binding ◽

Competitive Inhibitor ◽

Structural Basis ◽

Substrate Affinity ◽

Protein Homeostasis ◽

Auxiliary Subunit

AbstractN-terminal acetylation is one of the most common protein modifications in eukaryotes and is carried out by N-terminal acetyltransferases (NATs). It plays important roles in protein homeostasis, localization, and interactions and is linked to various human diseases. NatB, one of the major co-translationally active NATs, is composed of the catalytic subunit Naa20 and the auxiliary subunit Naa25, and acetylates about 20% of the proteome. Here we show that NatB substrate specificity and catalytic mechanism are conserved among eukaryotes, and that Naa20 alone is able to acetylate NatB substrates in vitro. We show that Naa25 increases the Naa20 substrate affinity, and identify residues important for peptide binding and acetylation activity. We present the first Naa20 crystal structure in complex with the competitive inhibitor CoA-Ac-MDEL. Our findings demonstrate how Naa20 binds its substrates in the absence of Naa25 and support prospective endeavors to derive specific NAT inhibitors for drug development.

Download Full-text

Structural basis of antigen recognition: crystal structure of duck egg lysozyme

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798317013730 ◽

2017 ◽

Vol 73 (11) ◽

pp. 910-920 ◽

Cited By ~ 2

Author(s):

David Brent Langley ◽

Ben Crossett ◽

Peter Schofield ◽

Jenny Jackson ◽

Mahdi Zeraati ◽

...

Keyword(s):

Crystal Structure ◽

Mass Spectrometry ◽

Binding Affinity ◽

Anas Platyrhynchos ◽

Antigen Recognition ◽

Pekin Duck ◽

Structural Basis ◽

Primary Sequence ◽

Duck Egg ◽

Differential Affinity

Duck egg lysozyme (DEL) is a widely used model antigen owing to its capacity to bind with differential affinity to anti-chicken egg lysozyme antibodies. However, no structures of DEL have so far been reported, and the situation had been complicated by the presence of multiple isoforms and conflicting reports of primary sequence. Here, the structures of two DEL isoforms from the eggs of the commonly used Pekin duck (Anas platyrhynchos) are reported. Using structural analyses in combination with mass spectrometry, non-ambiguous DEL primary sequences are reported. Furthermore, the structures and sequences determined here enable rationalization of the binding affinity of DEL for well documented landmark anti-lysozyme antibodies.

Download Full-text