A Binding Site Hotspot Map of the FKBP12–Rapamycin–FRB Ternary Complex by Photoaffinity Labeling and Mass Spectrometry-Based Proteomics

Structural characterization of small
molecule binding site hotspots
within the global proteome is uniquely enabled by photoaffinity labeling
(PAL) coupled with chemical enrichment and unbiased analysis by mass
spectrometry (MS). MS-based binding site maps provide structural resolution
of interaction sites in conjunction with identification of target
proteins. However, binding site hotspot mapping has been confined
to relatively simple small molecules to date; extension to more complex
compounds would enable the structural definition of new binding modes
in the proteome. Here, we extend PAL and MS methods to derive a binding
site hotspot map for the immunosuppressant rapamycin, a complex macrocyclic
natural product that forms a ternary complex with the proteins FKBP12
and FRB. Photo-rapamycin was developed as a diazirine-based PAL probe
for rapamycin, and the FKBP12–photo-rapamycin–FRB ternary
complex formed readily in vitro. Photoirradiation, digestion, and
MS analysis of the ternary complex revealed a McLafferty rearrangement
product of photo-rapamycin conjugated to specific surfaces on FKBP12
and FRB. Molecular modeling based on the binding site map revealed
two distinct conformations of complex-bound photo-rapamycin, providing
a 5.0 Å distance constraint between the conjugated residues and
the diazirine carbon and a 9.0 Å labeling radius for the diazirine
upon photoactivation. These measurements may be broadly useful in
the interpretation of binding site measurements from PAL. Thus, in
characterizing the ternary complex of photo-rapamycin by MS, we applied
binding site hotspot mapping to a macrocyclic natural product and
extracted precise structural measurements for interpretation of PAL
products that may enable the discovery of new binding sites in the
“undruggable” proteome.

全局蛋白质组内小分子结合位点热点的结构表征，唯有通过光亲和标记（photoaffinity labeling, PAL）结合化学富集与质谱（mass spectrometry, MS）无偏分析方可实现。基于质谱的结合位点图谱可在鉴定靶蛋白的同时，解析相互作用位点的结构分辨率。然而迄今为止，结合位点热点图谱的研究对象仅局限于相对简单的小分子；若将其拓展至更复杂的化合物，则有望实现蛋白质组内新型结合模式的结构定义。本研究将光亲和标记与质谱方法进行拓展，为免疫抑制剂雷帕霉素构建结合位点热点图谱。雷帕霉素是一种复杂的大环天然产物，可与FKBP12和FRB蛋白形成三元复合物。我们开发了基于双吖丙啶（diazirine）的光亲和标记探针光活化雷帕霉素（photo-rapamycin）用于雷帕霉素的研究，且FKBP12-光活化雷帕霉素-FRB三元复合物可在体外高效形成。对该三元复合物进行光照射、酶解与质谱分析后，发现光活化雷帕霉素的麦克拉弗蒂重排（McLafferty rearrangement）产物结合于FKBP12与FRB的特定表面。基于结合位点图谱的分子建模结果显示，结合于复合物的光活化雷帕霉素存在两种不同构象，给出了结合残基与双吖丙啶碳之间5.0 Å的距离约束，以及光活化后双吖丙啶的9.0 Å标记半径。这些测量结果可广泛用于解读光亲和标记实验得到的结合位点数据。因此，本研究通过质谱表征光活化雷帕霉素的三元复合物，将结合位点热点图谱拓展应用至大环天然产物，并提取出精确的结构测量数据，用于解读光亲和标记产物，有望助力发现“不可成药”蛋白质组中的新型结合位点。