Structural basis for L-isoaspartyl-containing protein recognition by the PCMTD1 cullin-RING E3 ubiquitin ligase

Pang, E. Z.; Zhao, B.; Flowers, C.; Oroudjeva, E.; Winter, J. B.; Pandey, V.; Sawaya, M. R.; Wohlschlegel, J.; Loo, J. A.; Rodriguez, J. A.; Clarke, S. G.

doi:10.1101/2025.05.21.654933

bioRxiv · 10.1101/2025.05.21.654933

Structural basis for L-isoaspartyl-containing protein recognition by the PCMTD1 cullin-RING E3 ubiquitin ligase

Pang, E. Z.·Zhao, B.·Flowers, C.·Oroudjeva, E.·Winter, J. B.·Pandey, V.·Sawaya, M. R.·Wohlschlegel, J.·Loo, J. A.·Rodriguez, J. A.·Clarke, S. G.

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Abstract

A major type of spontaneous protein damage that accumulates with age is the formation of kinked polypeptide chains with L-isoaspartyl residues. Mitigating this damage is necessary for maintaining proteome stability and prolonging organismal survival. While repair through methylation by PCMT1 has been previously shown to suppress L-isoaspartyl accumulation, we provide an additional mechanism for L-isoaspartyl maintenance through PCMTD1, a cullin-RING ligase (CRL). We combined cryo-EM, native mass spectrometry, and biochemical assays to provide insight on how the assembly and architecture of PCMTD1 in the context of a CRL complex fulfils this alternative mechanism. We show that the PCMTD1 CRL complex specifically binds L-isoaspartyl residues when bound to AdoMet. This work provides evidence for a growing class of E3 ubiquitin ligases that recognize spontaneous covalent modifications as potential substrates for ubiquitylation and subsequent proteasomal degradation. eTOC BlurbLimiting the accrual of L-isoaspartyl damaged proteins is essential during aging. While this is thought to be mediated solely by the repair activity of the protein, PCMT1, Pang et al. now demonstrate that a related protein, PCMTD1, functions as a cullin-RING ligase to selectively target L-isoaspartyl-damaged substrates for potential regulation by the ubiquitylation-proteosomal system. HighlightsO_LIAtomic cryo-EM structure of CRL5-PCMTD1 determined C_LIO_LIArchitecture of PCMTD1 when complexed as a CRL supports ubiquitylation activity C_LIO_LIPCMTD1 recognizes L-isoaspartyl residues as a recruitment motif for potential CRL activity C_LIO_LIRecognition of L-isoaspartyl residues is dependent on cofactor engagement C_LI

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Pang, E. Z., Zhao, B., Flowers, C., Oroudjeva, E., Winter, J. B., Pandey, V., Sawaya, M. R., Wohlschlegel, J., Loo, J. A., Rodriguez, J. A., Clarke, S. G.. 2025-05-21. Structural basis for L-isoaspartyl-containing protein recognition by the PCMTD1 cullin-RING E3 ubiquitin ligase. https://doi.org/10.1101/2025.05.21.654933

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