Vetrano Alice, Di Ianni Alessio, Di Fonte Nico, Dell'Orletta Gianluca, Reale Samantha, Daidone Isabella, Iacobucci Claudio
Department of Physical and Chemical Sciences, University of L'Aquila, Via Vetoio, L'Aquila, 67100, Italy.
Angew Chem Int Ed Engl. 2025 Jul 28;64(31):e202507348. doi: 10.1002/anie.202507348. Epub 2025 Jun 2.
Cross-linking mass spectrometry (XL-MS) has become a powerful tool in structural biology for investigating protein structure, dynamics, and interactomics. However, short-range cross-links, defined as those connecting residues fewer than 20 positions apart, have traditionally been considered less informative and largely overlooked, leaving significant data unexplored in a systematic manner. Here, we present a system-wide analysis of short-range cross-links, demonstrating their intrinsic correlation with protein secondary structure. We introduce the X-SPAN (Cross-link Structural Pattern Analyzer) software, which integrates publicly available XL-MS datasets from system-wide experiments with AlphaFold-predicted protein structures. Our analysis reveals distinct cross-linking patterns that reflect the spatial constraints imposed by secondary structural elements. Specifically, α-helices exhibit periodic cross-linking patterns consistent with their characteristic helical pitch, whereas coils and β-strands display nearly monotonic distributions. A context-dependent protein grammar reinforces short-range cross-link specificity. Short-range cross-links can enhance the statistical inference of secondary structures within integrative modeling workflows. Additionally, our work establishes a framework for benchmarking AlphaFold's local prediction accuracy and provides novel quality control criteria for XL-MS experiments. We anticipate that X-SPAN and our short-range cross-link database will serve as a valuable resource for exploring local secondary structure rearrangements and their potential roles in protein function and allosteric regulation.
交联质谱（XL-MS）已成为结构生物学中用于研究蛋白质结构、动力学和相互作用组学的强大工具。然而，短程交联（定义为连接相隔少于20个位置的残基的交联）传统上被认为信息量较少且大多被忽视，导致大量数据未得到系统探索。在此，我们对短程交联进行了全系统分析，证明了它们与蛋白质二级结构的内在相关性。我们引入了X-SPAN（交联结构模式分析仪）软件，该软件将全系统实验中公开可用的XL-MS数据集与AlphaFold预测的蛋白质结构整合在一起。我们的分析揭示了不同的交联模式，这些模式反映了二级结构元件所施加的空间限制。具体而言，α螺旋呈现出与其特征螺旋螺距一致的周期性交联模式，而无规卷曲和β链则表现出近乎单调的分布。一种依赖上下文的蛋白质语法增强了短程交联的特异性。短程交联可以增强整合建模工作流程中二级结构的统计推断。此外，我们的工作建立了一个用于评估AlphaFold局部预测准确性并为XL-MS实验提供新的质量控制标准的框架。我们预计X-SPAN和我们的短程交联数据库将成为探索局部二级结构重排及其在蛋白质功能和变构调节中潜在作用的宝贵资源。
