ABSTRACT
The nucleocapsid (N) protein of the SARS-CoV-2 virus, the causal agent of COVID-19, is a multifunction phosphoprotein that plays critical roles in the virus life cycle, including transcription and packaging of the viral RNA. To play such diverse roles, the N protein has two globular RNA-binding modules, the N- (NTD) and C-terminal (CTD) domains, which are connected by an intrinsically disordered region. Despite the wealth of structural data available for the isolated NTD and CTD, how these domains are arranged in the full-length protein and how the oligomerization of N influences its RNA-binding activity remains largely unclear. Herein, using experimental data from electron microscopy and biochemical/biophysical techniques combined with molecular modeling and molecular dynamics simulations, we show that, in the absence of RNA, the N protein formed structurally dynamic dimers, with the NTD and CTD arranged in extended conformations. However, in the presence of RNA, the N protein assumed a more compact conformation where the NTD and CTD are packed together. We also provided an octameric model for the full-length N bound to RNA that is consistent with electron microscopy images of the N protein in the presence of RNA. Together, our results shed new light on the dynamics and higher-order oligomeric structure of this versatile protein.
Subject(s)
Coronavirus Nucleocapsid Proteins , SARS-CoV-2 , COVID-19 , Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/metabolism , Humans , Microscopy, Electron , Molecular Dynamics Simulation , Nucleocapsid Proteins/chemistry , Nucleocapsid Proteins/metabolism , Phosphoproteins/metabolism , Protein Binding , RNA, Viral/genetics , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , SARS-CoV-2/metabolismABSTRACT
BACKGROUND: Biomolecular interactions that modulate biological processes occur mainly in cavities throughout the surface of biomolecular structures. In the data science era, structural biology has benefited from the increasing availability of biostructural data due to advances in structural determination and computational methods. In this scenario, data-intensive cavity analysis demands efficient scripting routines built on easily manipulated data structures. To fulfill this need, we developed pyKVFinder, a Python package to detect and characterize cavities in biomolecular structures for data science and automated pipelines. RESULTS: pyKVFinder efficiently detects cavities in biomolecular structures and computes their volume, area, depth and hydropathy, storing these cavity properties in NumPy arrays. Benefited from Python ecosystem interoperability and data structures, pyKVFinder can be integrated with third-party scientific packages and libraries for mathematical calculations, machine learning and 3D visualization in automated workflows. As proof of pyKVFinder's capabilities, we successfully identified and compared ADRP substrate-binding site of SARS-CoV-2 and a set of homologous proteins with pyKVFinder, showing its integrability with data science packages such as matplotlib, NGL Viewer, SciPy and Jupyter notebook. CONCLUSIONS: We introduce an efficient, highly versatile and easily integrable software for detecting and characterizing biomolecular cavities in data science applications and automated protocols. pyKVFinder facilitates biostructural data analysis with scripting routines in the Python ecosystem and can be building blocks for data science and drug design applications.
Subject(s)
COVID-19 , Data Science , Data Analysis , Ecosystem , Humans , SARS-CoV-2ABSTRACT
Lipases are water-soluble enzymes that catalyze the hydrolysis of lipids. Since lipids are mostly hydrophobic, lipase activity occurs preferentially at interfaces of aqueous and organic phases. In this work, we study the molecular mechanisms by which the Burkholderia cepacia lipase (BCL) is activated at interfaces of water with octane and with methyl caprylate (CAME). We show that BCL assumes very rapidly a preferential orientation at the interfaces, in which the active site is exposed to the organic phase. With BCL oriented to the interface, we compute the free energy of the aperture of the catalytic pocket using Adaptive Biasing Force MD simulations. The exposure to the organic phase promotes a clear stabilization of the open form of the catalytic pocket relative to the enzyme in water. This stabilization stems from the hydrophobicity of domains U1 and U2, which allows the penetration of organic solvents into the catalytic cleft impeding the closure of the pocket. Our results suggest that the structure and hydrophobicity of BCL are optimized for its activation in biphasic systems through the regulation of the accessibility of the catalytic pocket by, and for, hydrophobic substrates. The understanding of this mechanism may be useful for the design of proteins with targeted activation.