Computational design and molecular dynamics simulations suggest the mode of substrate binding in ceramide synthases

Zelnik, Iris Daphne; Mestre, Beatriz; Weinstein, Jonathan; Dingjan, Tamir; Izrailov, Stav; Ben-Dor, Shifra; Fleishman, Sarel J.; Futerman, Anthony H.

doi:10.1038/s41467-023-38047-x

Cited by 13 publications

(4 citation statements)

References 40 publications

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“…In this model, the substrates bind orthogonally, and the long-chain base would access the active site through a side entrance located near the membrane mid-point, where it would bind parallel to the membrane plane. This proposed substrate-binding arrangement is similar to that of small-molecule membrane-bound Oacyltransferases (MBOATs) and led to the suggestion that CerS may catalyze the direct transfer of the acyl chain from acyl-CoA to the long-chain base in a single step involving the two active site histidines 50 . In addition, while this manuscript was under review, the cryo-EM structure of yeast Lac1p bound to an acyl-CoA was reported.…”

Section: Discussionmentioning

confidence: 88%

“…This hydrocarbon chain lies against the conserved Trp318 of TM7 just beneath the active site. Mutation of the corresponding residue in CerS5 to an alanine has been previously shown to abolish enzymatic activity 50 . In contrast, introducing a phenylalanine at this position by a W318F mutation in CerS6 retained approximately wild-type levels of activity (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Recently, Zelnik et al postulated that CerS2, and ceramide synthases more generally, use a ternary complex reaction mechanism 50 . In this model, the substrates bind orthogonally, and the long-chain base would access the active site through a side entrance located near the membrane mid-point, where it would bind parallel to the membrane plane.…”

Section: Discussionmentioning

confidence: 99%

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Structural basis of the mechanism and inhibition of a human ceramide synthase

Pascoa,

Pike,

Tautermann

et al. 2023

Preprint

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Ceramides are bioactive sphingolipids that play pivotal roles in regulating cellular metabolism. Ceramides and dihydroceramides are synthesized by a family of six ceramide synthase enzymes (CerS), each with distinct specificity for the acyl-CoA substrate. Importantly, the acyl chain length plays a key role in determining the physiological function of ceramides, as well as their role in metabolic disease. Ceramide with an acyl chain length of 16 carbons (C16 ceramide) has been implicated in obesity, insulin resistance and liver disease, and the C16 ceramide-synthesizing CerS6 is regarded as an attractive drug target for obesity-associated disease. Despite their importance, the molecular mechanism underlying ceramide synthesis by CerS enzymes remains poorly understood. Here, we report cryo-electron microscopy structures of human CerS6, capturing covalent intermediate and product-bound states. These structures, together with biochemical characterization using intact protein and small molecule mass spectrometry, reveal that CerS catalysis proceeds via a ping-pong reaction mechanism involving a covalent acyl-enzyme intermediate. Notably, the product-bound structure was obtained upon reaction with the mycotoxin fumonisin B1, providing new insights into its inhibition of CerS. These results provide a framework for understanding the mechanisms of CerS function, selectivity, and inhibition, and open new directions for future drug discovery targeting the ceramide and sphingolipid pathways.

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Section: Discussionmentioning

confidence: 88%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Structural basis of the mechanism and inhibition of a human ceramide synthase

Pascoa,

Pike,

Tautermann

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…Further experimental analysis will be required to decide between these possibilities. In a companion paper, we demonstrate that mPROSS can also increase the stability and expression levels while retaining almost wild‐type activity levels in a human membrane enzyme based on its AlphaFold2 model structure (Zelnik et al, 2023 ). In that case, two homologous human ceramide synthases were subjected to modeling and design, and for one of these, a large increase in expression was observed while a few designs exhibited some decrease in activity at the highest mutation levels.…”

Section: Discussionmentioning

confidence: 99%

One‐shot design elevates functional expression levels of a voltage‐gated potassium channel

Weinstein,

Saikia,

Karbat

et al. 2024

Protein Science

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Membrane proteins play critical physiological roles as receptors, channels, pumps, and transporters. Despite their importance, however, low expression levels often hamper the experimental characterization of membrane proteins. We present an automated and web‐accessible design algorithm called mPROSS (https://mPROSS.weizmann.ac.il), which uses phylogenetic analysis and an atomistic potential, including an empirical lipophilicity scale, to improve native‐state energy. As a stringent test, we apply mPROSS to the Kv1.2–Kv2.1 paddle chimera voltage‐gated potassium channel. Four designs, encoding 9–26 mutations relative to the parental channel, were functional and maintained potassium‐selective permeation and voltage dependence in Xenopus oocytes with up to 14‐fold increase in whole‐cell current densities. Additionally, single‐channel recordings reveal no significant change in the channel‐opening probability nor in unitary conductance, indicating that functional expression levels increase without impacting the activity profile of individual channels. Our results suggest that the expression levels of other dynamic channels and receptors may be enhanced through one‐shot design calculations.

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Why do we study sphingolipids?

Futerman

2024

Pflugers Arch - Eur J Physiol

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Research on sphingolipids has proliferated exponentially over the past couple of decades, as exemplified in the findings reported at the International Leopoldina Symposium on Lipid Signaling held in Frankfurt in late 2023. Most researchers in the field study how sphingolipids function in regulating a variety of cellular processes and, in particular, how they are dysregulated in numerous human diseases; however, I now propose that we implement a more holistic research program in our study of sphingolipids, which embraces a sense of awe and wonder at the complexities and beauty of sphingolipids and of sphingolipid metabolism. I will outline the chemical complexity of sphingolipids, their modes of interaction within the lipid bilayer, and their biosynthetic pathways. I will then briefly touch upon the ability of current neo-Darwinian mechanisms to explain the emergence of both sphingolipids and of the complex pathways that generate them. Although such discussion is normally considered taboo in biological circles, I nevertheless submit that in-depth analysis of the minutiae of metabolic pathways, such as those of the sphingolipid biosynthetic pathway, raises challenges to current neo-Darwinian mechanisms that should not be shunned or ignored.

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Computational design and molecular dynamics simulations suggest the mode of substrate binding in ceramide synthases

Cited by 13 publications

References 40 publications

Structural basis of the mechanism and inhibition of a human ceramide synthase

Structural basis of the mechanism and inhibition of a human ceramide synthase

One‐shot design elevates functional expression levels of a voltage‐gated potassium channel

Why do we study sphingolipids?

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