Biology subjects

El Sahili, A.

Publications and source records attributed to El Sahili, A..

3 recordsLinked to original sources

bioRxiv2021

Structural basis for substrate recognition, ligation and activation by a hyperactive Asn peptide ligase from Viola yedoensis

Peptide asparaginyl ligases (PALs) belong to a limited class of enzymes from cyclotide-producing plants, that perform site-specific ligation reactions after a target peptide Asx (Asn/Asp) binds to the ligase active site. How PALs specifically recognize their polypeptide substrates has remained elusive especially at the prime binding side of the enzyme. Here we captured VyPAL2, a catalytically efficient PAL from Viola yedoensis, in an activated state, with and without a bound substrate. The bound structure shows one ligase with the N-terminal polypeptide tail from another ligase molecule trapped at its active site, revealing how Asx inserts in the enzymes S1 pocket and why a hydrophobic residue is required at the substrate P2 position. Beside illustrating the role played by P1 and P2 residues as primary anchors for the enzyme reaction, these results provide a mechanistic explanation for the role of the "Gatekeeper" residue at the surface of the S2 pocket, in shifting the non-prime portion of the substrate and, as a result, the activity towards either ligation or hydrolysis. These results detail the molecular events that occur during proenzyme maturation in the plant vacuolar compartment, suggest a mechanism for ligation, and will inform the design of peptide ligases with tailored specificities. One sentence summaryWe captured VyPAL2, a catalytically efficient plant peptide ligase with a bound substrate, providing the molecular basis for substrate recognition and ligation.

biochemistry↗

bioRxiv2021

Identification of an inhibitory pocket in falcilysin bound by chloroquine provides a new avenue for malaria drug development

Despite their widespread use, our understanding of how many antiparasitic drugs work remains limited. We used mass-spectrometry based cellular thermal shift assay (MS-CETSA) to identify possible protein targets of several malaria drugs and drug candidates. We found that falcilysin (FLN) is a common target for several quinoline drugs including chloroquine and mefloquine, as well as drug candidates MK-4815, MMV000848 and MMV665806. At pH 7.5, these compounds all inhibit FLN proteolytic activity with IC50 values ranging from 1.6 to 67.9 {micro}M. Their interaction with FLN was systematically probed by isothermal titration calorimetry and X-ray crystallography, revealing a shared hydrophobic pocket in the catalytic chamber of the enzyme. Characterization of transgenic cell lines with depleted FLN expression demonstrated statistically significant increases in susceptibility towards chloroquine, mefloquine, MK-4815 and MMV000848. Taken together, our findings point to a multimodal mechanism of action for several commonly used anti-malaria drugs. Importantly, a common allosteric pocket of FLN appears amenable to inhibition, providing a structural basis to guide the development of novel drugs against malaria.

microbiology↗

bioRxiv2020

Crystal structures of full length DENV4 NS2B-NS3 reveal the dynamic interaction between NS2B and NS3 upon binding to protease inhibitors

Flavivirus is a genus of emerging and re-emerging arboviruses which include many significant human pathogens. Non-structural protein 3 (NS3), a multifunctional protein with N-terminal protease and C-terminal helicase, is essential in viral replication. The NS3 protease together with NS2B cofactor is an attractive antiviral target. A construct with an artificial glycine linker connecting the NS2B cofactor and NS3 protease has been used for structural, biochemical and drug-screening studies. The effect of this linker on dynamics and enzymatic activity of the protease was studied by several biochemical and NMR methods but the findings remained inconclusive. Here, we designed constructs of NS2B cofactor joined to full length DENV4 NS3 in three different manners, namely bNS2B47NS3 (bivalent), eNS2B47NS3(enzymatically cleavable) and gNS2B47NS3 (glycine-rich G4SG4 linker). We report the first crystal structures of linked and unlinked full-length NS2B-NS3 enzyme in its free state and also in complex with Bovine Pancreatic Trypsin Inhibitor (BPTI). These structures demonstrate that the NS2B-NS3 protease mainly adopts a closed conformation. BPTI binding is not essential to but favors the closed conformation by interacting with both NS2B and NS3. The artificial linker between NS2B and NS3 tends to induce the open conformation and interfere with the protease activity. This negative impact on the enzyme structure and function is restricted to the protease domain as the ATPase activities of these constructs are not affected.

biochemistry↗