Ubiquitin-specific protease 11 structure in complex with an engineered substrate mimetic reveals a molecular feature for deubiquitination selectivity

Maurer, Sigrun K.; Mayer, Matthias P.; Ward, Stephanie J.; Boudjema, Sana; Halawa, Mohamed; Zhang, Jiatong; Caulton, Simon G.; Emsley, Jonas; Dreveny, Ingrid

doi:10.1016/j.jbc.2023.105300

Cited by 2 publications

References 67 publications

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Assessing the functional roles of coevolving PHD finger residues

Basu,

Subedi,

Tonelli

et al. 2024

Protein Science

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Although in silico folding based on coevolving residue constraints in the deep‐learning era has transformed protein structure prediction, the contributions of coevolving residues to protein folding, stability, and other functions in physical contexts remain to be clarified and experimentally validated. Herein, the PHD finger module, a well‐known histone reader with distinct subtypes containing subtype‐specific coevolving residues, was used as a model to experimentally assess the contributions of coevolving residues and to clarify their specific roles. The results of the assessment, including proteolysis and thermal unfolding of wildtype and mutant proteins, suggested that coevolving residues have varying contributions, despite their large in silico constraints. Residue positions with large constraints were found to contribute to stability in one subtype but not others. Computational sequence design and generative model‐based energy estimates of individual structures were also implemented to complement the experimental assessment. Sequence design and energy estimates distinguish coevolving residues that contribute to folding from those that do not. The results of proteolytic analysis of mutations at positions contributing to folding were consistent with those suggested by sequence design and energy estimation. Thus, we report a comprehensive assessment of the contributions of coevolving residues, as well as a strategy based on a combination of approaches that should enable detailed understanding of the residue contributions in other large protein families.

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Assessing the functional roles of coevolving PHD finger residues

Basu,

Subedi,

Tonelli

et al. 2024

Protein Science

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The structural and biophysical basis of substrate binding to the hydrophobic groove in Ubiquilin Sti1 domains

Onwunma,

Binsabaan,

Allen

et al. 2024

Preprint

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Ubiquilins are a family of cytosolic proteins that ferry ubiquitinated substrates to the proteasome for degradation. Recent work has demonstrated that Ubiquilins can also act as molecular chaperones, utilizing internal Sti1 domains to directly bind to hydrophobic sequences. Ubiquilins are associated with several neurodegenerative diseases with point mutations in UBQLN2 causing dominant, X-linked Amyotrophic Lateral Sclerosis (ALS). The molecular basis of Ubiquilin chaperone activity and how ALS mutations in the Sti1 domains affect Ubiquilin activity are poorly understood. This study presents the first crystal structure of the Sti1 domain from a fungal Ubiquilin homolog bound to a transmembrane domain (TMD). The structure reveals that two Sti1 domains form a head-to-head dimer, creating a hydrophobic cavity that accommodates two TMDs. Mapping the UBQLN2 sequence onto the structure shows that several ALS mutations are predicted to disrupt the hydrophobic groove. Using a newly developed competitive binding assay, we show that Ubiquilins preferentially bind to hydrophobic substrates with low helical propensity, motifs that are enriched in both substrates and in Ubiquilins. This study provides insights into the molecular and structural basis for Ubiquilin substrate binding, with broad implications for the role of the Sti1 domain in phase separation and ALS.

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Ubiquitin-specific protease 11 structure in complex with an engineered substrate mimetic reveals a molecular feature for deubiquitination selectivity

Cited by 2 publications

References 67 publications

Assessing the functional roles of coevolving PHD finger residues

Assessing the functional roles of coevolving PHD finger residues

The structural and biophysical basis of substrate binding to the hydrophobic groove in Ubiquilin Sti1 domains

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