Extended Data Fig. 3: Characterization of lactate and zinc binding to SENP1.
From: Lactate regulates cell cycle by remodelling the anaphase promoting complex
(a) 1H-15N TROSY HSQC spectrum of SENP1419–644 +/− zinc binding, highlighting all active site regions. (b) Intensity reduction in SENP1 resonances as a function of residue number after addition of 100 µM zinc to SENP1. (c) Structure of SENP1 (PDB ID 2IYC) color-coded as a heatmap according to peak intensity reduction caused by the addition of 100 µM zinc (cyan = no intensity reduction, red = maximum intensity reduction). (d) 1H-15N TROSY HSQC spectra of SENP1419–644 + 100 µM zinc and +/− 1 mM L-lactate, highlighting active site residues. (e) 1H-15N HSQC spectrum of human SENP1 catalytic domain comparing WT protein to C535S mutant. Spectral shifts differentiating WT and C535S SENP1 that match assignment to C535 in annotated spectra are highlighted. (f) MS determination and quantification of SENP1 cysteines that coordinate zinc. Method reports on stoichiometry of zinc binding with individual cysteines based on differential labelling with iodoacetamide (IAM)38. See Methods for details. (g) Quantification of zinc binding to human SENP1 cysteines by MS ± 1 mM lactate. Stoichiometry of zinc labelling of cysteines determined as described in Extended Data Fig. 3f, n = 5 independent samples. (h) 1H-15N HSQC spectrum of human WT SENP1 ± ZnSO4 in the presence or absence of 0.5 mM L-lactate, D-lactate, or pyruvate. Active site residues where L-lactate+zinc induced CSPs are observed are highlighted. (i) 1H-15N NMR spectra regions of WT SENP1 comparing L-lactate+zinc induced active site CSPs to D-lactate and pyruvate. (j) 1H-15N HSQC spectrum of human WT SENP1 ± 0.5 mM lactate. Regions of the spectrum where L-lactate+zinc induced CSPs in the active site are highlighted showing none of these CSPs occur in the absence of zinc. (one-way ANOVA for multiple comparisons in g. ANOVA analyses were subjected to Bonferroni’s post hoc test). Data are mean ± s.e.m.
