Biology subjects

De Munck, S.

Publications and source records attributed to De Munck, S..

3 recordsLinked to original sources

bioRxiv2024

Reanalysis of cryo-EM data reveals ALK-cytokine assemblies with distinct stoichiometries

Activation of Anaplastic lymphoma kinase (ALK) and leukocyte tyrosine kinase (LTK) by their cognate cytokines ALKAL2 and ALKAL1 play important roles in development, metabolism, and cancer. Recent structural studies revealed ALK/LTK-cytokine assemblies with distinct stoichiometries. Structures of ALK-ALKAL2 and LTK-ALKAL1 complexes with 2:1 stoichiometry determined by X-ray crystallography (De Munck et al. Nature, 2021) contrasted the 2:2 ALK-ALKAL2 complexes determined by cryo-EM (Reshetnyak et al., Nature, 2021) and X-ray crystallography (Li et al., Nature, 2021). Here, we show based on reanalysis of the cryo-EM data deposited in EMPIAR- 10930 that over half of the ALK-ALKAL2 particles in the dataset are classified into 2D classes obeying a 2:1 stoichiometry besides the originally reported structure displaying 2:2 stoichiometry. Unlike particles representing the 2:2 ALK-ALKAL2 complex, particles for the 2:1 ALK-ALKAL2 complex suffer severely from preferred orientations that resulted in cryo-EM maps displaying strong anisotropy. Here, we show that extensive particle orientation rebalancing in cryoSPARC followed by 3D refinement with Blush regularization in RELION constitutes an effective strategy for avoiding map artefacts relating to preferred particle orientations and report a 3D reconstruction of the 2:1 ALK-ALKAL2 complex to 3.2 [A] resolution from EMPIAR-10930. This new cryo-EM structure together with the crystal structures of ALK-ALKAL2 and LTK-ALKAL1 complexes with 2:1 stoichiometry reconciles a common receptor dimerization mode for ALK and LTK and provides direct evidence for the presence of an ALK-ALKAL2 complex with 2:1 stoichiometry next to the reported 2:2 stoichiometric assembly in the EMPIAR-10930 dataset. Finally, our analysis emphasizes the importance of public deposition of raw cryo-EM data to allow reanalysis and interpretation.

molecular biology↗

bioRxiv2022

Improving de novo Protein Binder Design with Deep Learning

We explore the improvement of energy-based protein binder design using deep learning. We find that using AlphaFold2 or RoseTTAFold to assess the probability that a designed sequence adopts the designed monomer structure, and the probability that this structure binds the target as designed, increases design success rates nearly 10-fold. We find further that sequence design using ProteinMPNN rather than Rosetta considerably increases computational efficiency.

bioengineering↗

bioRxiv2020

Distinct EH domains of the endocytic TPLATE complex confer lipid and protein binding.

Clathrin-mediated endocytosis (CME) is the gatekeeper of the plasma membrane. In contrast to animals and yeasts, CME in plants depends on the TPLATE complex (TPC), an evolutionary ancient adaptor complex. The mechanistic contribution of the individual TPC subunits to plant CME remains however elusive. In this study, we used a multidisciplinary approach to elucidate the structural and functional roles of the evolutionary conserved N-terminal Eps15 homology (EH) domains of the TPC subunit AtEH1/Pan1. By integrating high-resolution structural information obtained by X-ray crystallography and NMR spectroscopy with all-atom molecular dynamics simulations, we provide structural insight into the function of both EH domains. Whereas one EH domain binds negatively charged PI(4,5)P2 lipids, unbiased peptidome profiling by mass-spectrometry revealed that the other EH domain interacts with the double N-terminal NPF motif of a novel TPC interactor, the integral membrane protein Secretory Carrier Membrane Protein 5 (SCAMP5). Furthermore, we show that AtEH/Pan1 proteins control the internalization of SCAMP5 via this double NPF peptide interaction motif. Collectively, our structural and functional studies reveal distinct but complementary roles of the EH domains of AtEH/Pan1 have in plant CME and connect the internalization of SCAMP5 to the TPLATE complex.

plant biology↗