The Effect Of Protein-Protein Association On The Dynamics And Thermodynamics Of SARS-CoV-2 RBD And Human ACE2

The pandemic of a new coronavirus SARS-CoV-2 has brought a serious impact on human health and global economy,and thus has been attracting the extensive attention of the scientific researchers in various fields.In order to investigate the mechanism of SARS-CoV-2 entry into the human cells,many structures of the partial or complete viral spike proteins both alone in complex with the human cell-surface receptor,angiotensin-converting enzyme 2（ACE2）,have been resolved;however,all these structures are static and hence cannot directly reflect the dynamic process of viral binding and entry,nor can they uncover the effects of protein-protein association on the dynamics,thermodynamics,and the functions of the binding partners.Consequently,here we took the receptor binding domain（RBD）on the spike protein of SARS-CoV-2and its receptor ACE2 as the research objects,and performed the multiple-replica molecular dynamics（MD）simulations on the structures of RBD-ACE2 complex（bound state）and the unbound/free structures of RBD and ACE2,respectively to obtain sufficient enough conformational sampling of them,with the total simulation time of 1ms for each system;subsequently,On the basis of the obtained concatenated equilibrium MD trajectories,the effects of protein-protein binding on the dynamic and thermodynamic behaviors of RBD and ACE2 were investigated through a series of comparative analyses in terms of the dynamic properties,principal component,and reconstruction and comparison of free energy landscapes.The results show that the association between ACE2 and RBD leads to i)intensified structural fluctuations,increased global conformational flexibility and conformational entropy,and reduced thermal stability of ACE2;ii)damped structural fluctuations,increased global conformational rigidity and the thermal stability,reduced conformational entropy,and increased difficulty in kinetics of conformational conversion between the two main states of RBD;iii)reversed largest-amplitude collective motion modes of ACE2 and RBD when compared to those in the free states.Therefore,it can be considered that the association between RBD and ACE2 could lead to the opposite changing trends in the dynamic and thermodynamic behaviors of the two binding partners.Based on our results,in conjunction with published experimental data related to the structural and functional properties of RBD and ACE2,it is reasonable to speculate that the increased stability of RBD upon binding to ACE2 could effectively maintain the standing state（or open state）of a single S1 subunit in the SARS-CoV-2 spike trimer,which in turn is conducive to the recognition and binding of the other RBDs by ACE2 and hence promotes the opening of all S1 subunits.On the other hand,the occurrence of the conformational transition event of RBD induced by ACE2 binding is very likely to facilitate the exposure of S2’ proteolytic cleavage site,thus allowing for the cleavage by the proteinase so as to trigger the transition of the spike protein from the pre-fusion conformation to the post-fusion conformation.The increased conformational flexibility of ACE2 upon RBD binding,in conjunciton of the observed closure of the substrate-binding groove of ACE2 upon RBD binding,will increase the catalytic activity and substrate-binding affinity of ACE2.Since the substrates and products of ACE2 paticipate in regulating the pathways of the cardiovascular system,immune response,and inflammation,the changes in the catalytic function of ACE2 caused by SARS-CoV-2 infection are very likely to be related to the observed symptoms of COVID-19 patients.This study sheds light on the structure-dynamics-function relationships of both RBD and ACE2 at the atomic level,thus facilitating an in-depth understanding of the cell entry mechanism of SARS-CoV-2 and of the pathogenic mechanism of COVID-19.