A Study Of New Methods For Target Protein Structure Regulation By Mass Spectrometry

Dynamic interaction analysis of biological macromolecules such as proteins and nucleic acids is of great significance for in-depth understanding of complex life processes and targeted drug development.Fine protein structure characterization is essential for probing interaction details,understanding protein function regulation,and targeted drug design.Based on cryo-electron microscopy and artificial intelligence technology,remarkable progress has been made in the resolution of static protein structures in recent years.However,these technologies are difficult to obtain accurate dynamic protein structure information.Proteomics analysis based on structural mass spectrometry has the characteristics of high throughput,high sensitivity and high accuracy,and has become a powerful tool to characterize the dynamic structure,interaction and function of proteins.However,a single structural mass spectrometry technique can only provide scattered and single dynamic structural information of proteins.In this work,we mainly developed new methods for protein complex structure characterization by integrating multiple mass spectrometry techniques to achieve a deep and fine characterization of protein dynamic structure.This method can provide the dynamic structure information of multi-dimensional proteins in the interaction characterization of small molecular-protein complexes and RNA-protein complexes,which proves that the dynamic structure information obtained by different mass spectrometry methods has good consistency and complementarity,and has great application prospects in the analysis of disease mechanisms and the development of targeted drugs.This study mainly includes two parts:(1)Integration of lysine reactivity profiling mass spectrometry and native mass spectrometry to detect inhibitor-induced allosteric activation of CDK12/CDK13-cyclin K dissociationThe rational design and development of effective inhibitors for cyclin-dependent kinases 12 and 13(CDK12 and CDK13)largely dependent on the understanding of the dynamic inhibition conformations,but difficult to be achieved by conventional characterization tools.Herein,we integrate the structural mass spectrometry methods of lysine reactivity profiling(LRP)and native MS(n MS)to systematically interrogate the dynamic molecular interactions of CDK12/CDK13-cyclin K(Cyc K)complexes under the modulation of small-molecular inhibitors.LRP can provide essential structure insights including inhibitor binding pocket,binding strength,interfacial molecular details,and dynamic conformation changes,while n MS can provide information on the overall protein assembly.We find the inhibitor SR-4835 binding can greatly weaken the CDK12/CDK13-Cyc K interactions in an unusual allosteric activation way,providing a novel alternative for the kinase activity inhibition.Our results underscore the great potential of LRP combination with n MS for the evaluation and rational design of effective kinase inhibitors at molecular level.(2)Integration lysine reactivity profiling mass spectrometry and peroxide-free photochemical oxidation of proteins to probe the molecular mechanism of RIG-I recognition of viral RNARetinoic acid-inducible gene I(RIG-I)is a key immune receptor that recognizes viral RNA and activates interferon-mediated antiviral responses.However,the long Linker has hindered the crystal structure resolution of full-length human RIG-I containing the CARDs region.The molecular description of the RIG-I activation pathway in humans remains speculative.In this study,we integrated LRP and peroxide-free photochemical oxidation of proteins(PPOP)to probe the molecular mechanism of RNA recognition by human RIG-I.LRP can provide information on lysine reactivity,and PPOP can provide information on the other nine amino acids.The complementary study of the two methods can provide more comprehensive structural information.We further demonstrate that human RIG-I maintains its self-inhibitory conformation through HEL2i-CARD2 interaction in the initial state,when the Pincer-attached CTD is in the unfolded state.After RNA binding,the CARDs unfold,and the Pincer-attached CTD folds to form a protein network that wraps the RNA.Taken together,our data provide more views of the conformational changes in RIG-I activation in solution and provide new evidence on the molecular mechanism of RIG-I activation,highlighting the great potential of LRP and PPOP for the molecular level assessment of protein-RNA interactions,which can contribute to antiviral therapy as well as the development of RIG-I agonists.