Hastings Renee, Aditham Arjun K, DelRosso Nicole, Suzuki Peter H, Fordyce Polly M
Biophysics Program, Stanford University, Stanford, CA, USA.
Department of Bioengineering, Stanford University, Stanford, CA, USA.
Nat Commun. 2025 Jan 14;16(1):636. doi: 10.1038/s41467-024-55672-2.
Understanding how proteins discriminate between preferred and non-preferred ligands ('selectivity') is essential for predicting biological function and a central goal of protein engineering efforts, yet the biophysical mechanisms underpinning selectivity remain poorly understood. Towards this end, we study how variants of the promiscuous transcription factor (TF) MAX (H. sapiens) alter DNA specificity and selectivity, yielding >1700 Ks and >500 rate constants in complex with multiple DNA sequences. Twenty-two of the 240 assayed MAX point mutations enhance selectivity, yet none of these mutations occur at residues that contact nucleotides in published structures. By applying thermodynamic and kinetic models to these results and previous observations for the highly similar yet far more selective TF Pho4 (S. cerevisiae), we find that these mutations enhance selectivity by altering partitioning between or affinity within conformations with different intrinsic selectivity, providing a mechanistic basis for allosteric modulation of ligand selectivity. These results highlight the importance of conformational heterogeneity in determining sequence selectivity and can guide future efforts to engineer selective proteins.
理解蛋白质如何区分优先配体和非优先配体（“选择性”）对于预测生物学功能至关重要，也是蛋白质工程研究的核心目标，然而，支撑选择性的生物物理机制仍知之甚少。为此，我们研究了混杂转录因子（TF）MAX（智人）的变体如何改变DNA特异性和选择性，在与多个DNA序列形成复合物时产生了超过1700个解离常数（Ks）和超过500个速率常数。在检测的240个MAX点突变中，有22个增强了选择性，但这些突变均未发生在已发表结构中与核苷酸接触的残基上。通过将热力学和动力学模型应用于这些结果以及之前对高度相似但选择性强得多的TF Pho4（酿酒酵母）的观察，我们发现这些突变通过改变具有不同内在选择性的构象之间的分配或亲和力来增强选择性，为配体选择性的变构调节提供了机制基础。这些结果突出了构象异质性在确定序列选择性中的重要性，并可为未来设计选择性蛋白质的工作提供指导。
