D614G mutation in the SARS-CoV-2 spike protein enhances viral fitness by desensitizing it to temperature-dependent denaturation

Abstract

The D614G mutation in the spike protein of SARS-CoV-2 alters the fitness of the virus, leading to the dominant form observed in the COVID-19 pandemic. However, the molecular basis of the mechanism by which this mutation enhances fitness is not clear. Here we demonstrated by cryo-electron microscopy that the D614G mutation resulted in increased propensity of multiple receptor-binding domains (RBDs) in an upward conformation poised for host receptor binding. Multiple substates within the one RBD-up or two RBD-up conformational space were determined. According to negative staining electron microscopy, differential scanning calorimetry, and differential scanning fluorimetry, the most significant impact of the mutation lies in its ability to eliminate the unusual cold-induced unfolding characteristics and to significantly increase the thermal stability under physiological pH. The D614G spike variant also exhibited exceptional long-term stability when stored at 37 °C for up to 2 months. Our findings shed light on how the D614G mutation enhances the infectivity of SARS-CoV-2 through a stabilizing mutation and suggest an approach for better design of spike protein-based conjugates for vaccine development. The D614G mutation in the spike protein of SARS-CoV-2 alters the fitness of the virus, leading to the dominant form observed in the COVID-19 pandemic. However, the molecular basis of the mechanism by which this mutation enhances fitness is not clear. Here we demonstrated by cryo-electron microscopy that the D614G mutation resulted in increased propensity of multiple receptor-binding domains (RBDs) in an upward conformation poised for host receptor binding. Multiple substates within the one RBD-up or two RBD-up conformational space were determined. According to negative staining electron microscopy, differential scanning calorimetry, and differential scanning fluorimetry, the most significant impact of the mutation lies in its ability to eliminate the unusual cold-induced unfolding characteristics and to significantly increase the thermal stability under physiological pH. The D614G spike variant also exhibited exceptional long-term stability when stored at 37 °C for up to 2 months. Our findings shed light on how the D614G mutation enhances the infectivity of SARS-CoV-2 through a stabilizing mutation and suggest an approach for better design of spike protein-based conjugates for vaccine development. The COVID-19 (coronavirus disease 2019) pandemic is caused by the infection of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) (1Zhu N. Zhang D. Wang W. Li X. Yang B. Song J. Zhao X. Huang B. Shi W. Lu R. Niu P. Zhan F. Ma X. Wang D. Xu W. et al.A novel coronavirus from patients with pneumonia in China, 2019.N. Engl. J. Med. 2020; 382: 727-733Crossref PubMed Scopus (17405) Google Scholar). Early bioinformatic analysis of the reported genome sequences of SARS-CoV-2 revealed the emergence of a prominent pairwise linkage disequilibrium between three nucleotide changes since mid-February 2020, namely nt3037 (C > T), nt14408 (C > T), and nt23403 (A > G). The last mutation corresponds to a missense D614G mutation in the spike (S) protein (2Yang H.-C. Chen C.-H. Wang J.-H. Liao H.-C. Yang C.-T. Chen C.-W. Lin Y.-C. Kao C.-H. Lu M.-Y.J. Liao J.C. Analysis of genomic distributions of SARS-CoV-2 reveals a dominant strain type with strong allelic associations.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 30679-30686Crossref PubMed Scopus (48) Google Scholar). The G clade, which harbors the aforementioned three nucleotide changes and the nt241 (C > T) mutation, relative to the original Wuhan form (hereafter wild type, WT), became the dominant form of the COVID-19 pandemic since the summer of 2020 (https://www.gisaid.org/). The enhanced infectivity of the D614G variant is observed in cell cultures as well as animal models (3Plante J.A. Liu Y. Liu J. Xia H. Johnson B.A. Lokugamage K.G. Zhang X. Muruato A.E. Zou J. Fontes-Garfias C.R. Mirchandani D. Scharton D. Bilello J.P. Ku Z. An Z. et al.Spike mutation D614G alters SARS-CoV-2 fitness.Nature. 2021; 592: 116-121Crossref PubMed Scopus (909) Google Scholar). The SARS-CoV-2 S protein (hereafter S protein) is responsible for host recognition and viral entry. The S protein binds to the receptor, angiotensin converting enzyme 2 (ACE2), through an upward open RBD conformation (RBD-up); a downward closed RBD conformation (RBD-down) sequesters its receptor-binding motif (RBM) from receptor binding, rendering such a conformation inactive (4Wrapp D. Wang N. Corbett K.S. Goldsmith J.A. Hsieh C.L. Abiona O. Graham B.S. McLellan J.S. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation.Science. 2020; 367: 1260-1263Crossref PubMed Scopus (84) Google Scholar, 5Walls A.C. Park Y.J. Tortorici M.A. Wall A. McGuire A.T. Veesler D. Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein.Cell. 2020; 181: 281-292.e6Abstract Full Text Full Text PDF PubMed Scopus (5486) Google Scholar). Several studies on COVID-19 convalescent sera have identified multiple antibodies that competitively bind to the RBM, and in doing so prevent host receptor ACE2 binding, thereby achieving neutralizing activities (6Piccoli L. Park Y.J. Tortorici M.A. Czudnochowski N. Walls A.C. Beltramello M. Silacci-Fregni C. Pinto D. Rosen L.E. Bowen J.E. Acton O.J. Jaconi S. Guarino B. Minola A. Zatta F. et al.Mapping neutralizing and immunodominant sites on the SARS-CoV-2 spike receptor-binding domain by structure-guided high-resolution serology.Cell. 2020; 183: 1024-1042.e21Abstract Full Text Full Text PDF PubMed Scopus (766) Google Scholar, 7Liu L. Wang P. Nair M.S. Yu J. Rapp M. Wang Q. Luo Y. Chan J.F. Sahi V. Figueroa A. Guo X.V. Cerutti G. Bimela J. Gorman J. Zhou T. et al.Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike.Nature. 2020; 584: 450-456Crossref PubMed Scopus (892) Google Scholar, 8Robbiani D.F. Gaebler C. Muecksch F. Lorenzi J.C.C. Wang Z. Cho A. Agudelo M. Barnes C.O. Gazumyan A. Finkin S. Hagglof T. Oliveira T.Y. Viant C. Hurley A. Hoffmann H.H. et al.Convergent antibody responses to SARS-CoV-2 in convalescent individuals.Nature. 2020; 584: 437-442Crossref PubMed Scopus (1169) Google Scholar, 9Barnes C.O. Jette C.A. Abernathy M.E. Dam K.A. Esswein S.R. Gristick H.B. Malyutin A.G. Sharaf N.G. Huey-Tubman K.E. Lee Y.E. Robbiani D.F. Nussenzweig M.C. West Jr., A.P. Bjorkman P.J. SARS-CoV-2 neutralizing antibody structures inform therapeutic strategies.Nature. 2020; 588: 682-687Crossref PubMed Scopus (823) Google Scholar, 10Cao Y. Su B. Guo X. Sun W. Deng Y. Bao L. Zhu Q. Zhang X. Zheng Y. Geng C. Chai X. He R. Li X. Lv Q. Zhu H. et al.Potent neutralizing antibodies against SARS-CoV-2 identified by high-throughput single-cell sequencing of convalescent patients' B cells.Cell. 2020; 182: 73-84.e16Abstract Full Text Full Text PDF PubMed Scopus (788) Google Scholar). Despite the large size, the S protein is marginally stable over a narrow range of temperatures. Incubation of the recombinant SARS-CoV-2 S protein at 4 °C leads to significant unfolding within 24 h. The morphological change of cold denaturation resembles that after a brief heat shock at 50 to 60 °C (11Xiong X. Qu K. Ciazynska K.A. Hosmillo M. Carter A.P. Ebrahimi S. Ke Z. Scheres S.H.W. Bergamaschi L. Grice G.L. Zhang Y. Collaboration C.-N.C.-B. Nathan J.A. Baker S. James L.C. et al.A thermostable, closed SARS-CoV-2 spike protein trimer.Nat. Struct. Mol. Biol. 2020; 27: 934-941Crossref PubMed Scopus (162) Google Scholar, 12Edwards R.J. Mansouri K. Stalls V. Manne K. Watts B. Parks R. Janowska K. Gobeil S.M.C. Kopp M. Li D. Lu X. Deyton M. Oguin T.H. O.r. Sprenz J. Williams W. et al.Cold sensitivity of the SARS-CoV-2 spike ectodomain.Nat. Struct. Mol. Biol. 2020; 28: 128-131Crossref Scopus (43) Google Scholar). As a vaccine candidate for mitigating COVID-19, the sensitivity of the recombinant SARS-CoV-2 S protein to cold- and heat-induced unfolding is a major concern. Here, we demonstrated the structural dynamics of the D614G variant spike protein (hereafter S-D614G) by using cryo-EM with the aid of three-dimensional variability analysis (3DVA) (13Punjani A. Fleet D.J. 3D variability analysis: Resolving continuous flexibility and discrete heterogeneity from single particle cryo-EM.J. Struct. Biol. 2021; 213: 107702Crossref PubMed Scopus (276) Google Scholar). Compared with the wild-type S (hereafter S-D614), the increased propensity of S-D614G in the RBD-up conformation implicated the facilitation of the binding to the receptor ACE2. We further conducted the negative staining electronic microscopy (NSEM) analysis coupled with differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF) to reveal that the D614G mutation eliminates the cold sensitivity of the original D614 and confers the resistance to the high temperature. Our study shows that the S protein with the D614G mutation is more thermally stable than S-D614, suggesting that S-D614G will be a new candidate for vaccine development. Using cryo-EM single particle reconstruction aided by 3DVA analysis (13Punjani A. Fleet D.J. 3D variability analysis: Resolving continuous flexibility and discrete heterogeneity from single particle cryo-EM.J. Struct. Biol. 2021; 213: 107702Crossref PubMed Scopus (276) Google Scholar, 14Yang T.J. Yu P.Y. Chang Y.C. Liang K.H. Tso H.C. Ho M.R. Chen W.Y. Lin H.T. Wu H.C. Hsu S.-T.D. Effect of SARS-CoV-2 B.1.1.7 mutations on spike protein structure and function.Nat. Struct. Mol. Biol. 2021; 28: 731-739Crossref PubMed Scopus (85) Google Scholar), we identified five distinct but equally populated clusters of conformations of S-D614G with varying degrees of RBD-up populations (Fig. 1, Fig. S1 and Table S1). Collectively, 62% of the total population of S-D614G had one RBD in an up conformation (one RBD-up), including three distinct substates that separated the RBD conformations, and 38% of the population had two RBDs in an up conformation (two RBD-up) with two different substates. The three distinct RBD-up substrates in the one RBD-up class showed a 39 to 52° upward rotation with respect to the hinge defined as the Cα atom of the beginning of RBD, i.e., residue 330 (Fig. the two distinct RBD-up substates in the two RBD-up class showed a to upward to S-D614, we not conformation in in with the study of a the domain D.J. A.G. C. A. Xu P. S.R. The of the D614G on the structure of the spike of Natl. Acad. Sci. U. S. A. 2021; PubMed Scopus Google Scholar), but from the S-D614G that significant of conformation J. Y. T. Lu J. H. Jr., S. Zhu H. Yang W. P. Chen B. impact on SARS-CoV-2 spike protein by D614G 2021; PubMed Scopus Google Scholar, Z. J. Qu K. M. V. L. T. J. B. Lu J. X. Scheres S.H.W. et and distributions of SARS-CoV-2 spike on 2020; 588: PubMed Scopus Google Scholar). studies reported cryo-EM structures of S-D614G with or the mutations the mutation which showed one RBD-up or conformations R. Zhang Y. Li Y. F. Guo Y. Xia L. X. X. Zhou Q. basis for the different of the spike protein of SARS-CoV-2 in with 2021; PubMed Scopus Google Scholar, Janowska K. S. Mansouri K. Parks R. Manne K. Stalls V. Kopp R. R.J. P. D614G mutation alters SARS-CoV-2 spike conformation and enhances at the 2021; Full Text Full Text PDF PubMed Scopus Google Scholar). Our study is one of the in to et D.J. A.G. C. A. Xu P. S.R. The of the D614G on the structure of the spike of Natl. Acad. Sci. U. S. A. 2021; PubMed Scopus Google Scholar), that reported the two RBD-up et L. Wang X. K.E. C. Wang Y. A. A. C. K. J.E. et and analysis of the D614G SARS-CoV-2 spike protein 2020; 183: Full Text Full Text PDF PubMed Scopus Google reported an RBD-up conformation of the of the RBDs is of reported S-D614G is the increased propensity to the RBD-up conformations, and the two RBD-up conformations have not reported for the upward conformation of the RBD is the of host receptor ACE2 binding, the increased population of the RBD-up conformations with more conformational S-D614G is to enhanced binding to the receptor ACE2 S. Zhang Y. H. K. K. K. S. T. Y. T. K. SARS-CoV-2 D614G spike mutation with enhanced 2021; PubMed Scopus Google Scholar). et L. Wang X. K.E. C. Wang Y. A. A. C. K. J.E. et and analysis of the D614G SARS-CoV-2 spike protein 2020; 183: Full Text Full Text PDF PubMed Scopus Google that the D614G mutation the between D614 of one and of the thereby leading to increase dynamics of the S protein and the of between the RBD-up and However, cryo-EM structure of S-D614G showed structural in the of the mutation with respect to of the of the reported including the reported structures of (4Wrapp D. Wang N. Corbett K.S. Goldsmith J.A. Hsieh C.L. Abiona O. Graham B.S. McLellan J.S. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation.Science. 2020; 367: 1260-1263Crossref PubMed Scopus (84) Google Scholar, 5Walls A.C. Park Y.J. Tortorici M.A. Wall A. McGuire A.T. Veesler D. Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein.Cell. 2020; 181: 281-292.e6Abstract Full Text Full Text PDF PubMed Scopus (5486) Google Scholar), the form of T. Y. Gorman J. Rapp M.A. Cerutti G. A. A. Wang Bimela J. Shi W. et structures of SARS-CoV-2 spike and with ACE2 reveal a to of receptor-binding 2020; 28: Full Text Full Text PDF PubMed Scopus Google Scholar), the and variant C.L. Goldsmith J.A. H.C. K. D. Lee A.G. Liu Y. Johnson J. et design of SARS-CoV-2 2020; PubMed Scopus Google Scholar), and the that a more open conformation A.G. D.J. Xu P. C. S.R. SARS-CoV-2 and spike structures inform on and Struct. Mol. Biol. 2020; 27: PubMed Scopus Google Scholar). et D.J. A.G. C. A. Xu P. S.R. The of the D614G on the structure of the spike of Natl. Acad. Sci. U. S. A. 2021; PubMed Scopus Google that the of a by D614 of one and or the to the D614G mutation leads to thereby the dynamics of S-D614G and more populated RBD-up conformations, a that be by cryo-EM structures (Fig. the molecular basis of how the D614G mutation change the conformation and dynamics of the RBDs to be that the structure of S-D614G is not significantly by the D614G mutation under we to the impact of the D614G mutation on the thermal stability over a range of that the S protein is to cold were using and which were the at 37 and at (Fig. We negative electron microscopy (NSEM) on and S-D614G on and on after continuous at 37 °C and 4 with the R.J. Mansouri K. Stalls V. Manne K. Watts B. Parks R. Janowska K. Gobeil S.M.C. Kopp M. Li D. Lu X. Deyton M. Oguin T.H. O.r. Sprenz J. Williams W. et al.Cold sensitivity of the SARS-CoV-2 spike ectodomain.Nat. Struct. Mol. Biol. 2020; 28: 128-131Crossref Scopus (43) Google Scholar), stable at 37 °C for morphological significant unfolding observed after at 4 °C for (Fig. analysis showed that the at 37 °C and 4 °C resulted in and of 2 and Table S-D614G not significant unfolding after the cold the at 37 °C and 4 °C resulted in and of 2 and after 2 of at 37 S-D614G to analysis (Fig. to heat-induced denaturation of at 50 °C and 60 exhibited significant unfolding at of heat shock at 50 °C and 60 in and of the heat shock resulted in and of for exhibited more in the of in the of heat at 50 °C for (Fig. that the significant in the of after cold heat the 3D of and S-D614G were at the of in Fig. suggesting that the unfolding of the S protein an in different of populations after the further analysis with more for S-D614G a with an of °C (Fig. particle of S-D614G as a of temperature. were at the as the for to The of in were with respect to the of The to the of the within the under the between 50 and 60 were 3D of the from the particle to the We further the thermal unfolding of and S-D614G at by and S-D614G exhibited two and a for the at However, S-D614G exhibited a for the with that of the total of unfolding of S-D614G than that for (Fig. and Table The of the of S-D614G reported R.J. Mansouri K. Stalls V. Manne K. Watts B. Parks R. Janowska K. Gobeil S.M.C. Kopp M. Li D. Lu X. Deyton M. Oguin T.H. O.r. Sprenz J. Williams W. et al.Cold sensitivity of the SARS-CoV-2 spike ectodomain.Nat. Struct. Mol. Biol. 2020; 28: 128-131Crossref Scopus (43) Google Scholar, T. Y. Gorman J. Rapp M.A. Cerutti G. A. A. Wang Bimela J. Shi W. et structures of SARS-CoV-2 spike and with ACE2 reveal a to of receptor-binding 2020; 28: Full Text Full Text PDF PubMed Scopus Google Scholar), and that the in this be to at T. Y. Gorman J. Rapp M.A. Cerutti G. A. A. Wang Bimela J. Shi W. et structures of SARS-CoV-2 spike and with ACE2 reveal a to of receptor-binding 2020; 28: Full Text Full Text PDF PubMed Scopus Google Scholar). the for after at 4 °C for that of S-D614G in with long-term at 37 °C not significantly (Fig. at 4 °C for long-term is to be more than at 37 this not to S-D614G not to cold denaturation and is more heat shock The of the D614G mutation most under to as demonstrated by of and S-D614G over a range of (Fig. exhibited multiple between 4 and as reported R.J. Mansouri K. Stalls V. Manne K. Watts B. Parks R. Janowska K. Gobeil S.M.C. Kopp M. Li D. Lu X. Deyton M. Oguin T.H. O.r. Sprenz J. Williams W. et al.Cold sensitivity of the SARS-CoV-2 spike ectodomain.Nat. Struct. Mol. Biol. 2020; 28: 128-131Crossref Scopus (43) Google Scholar, T. Y. Gorman J. Rapp M.A. Cerutti G. A. A. Wang Bimela J. Shi W. et structures of SARS-CoV-2 spike and with ACE2 reveal a to of receptor-binding 2020; 28: Full Text Full Text PDF PubMed Scopus Google Scholar). The the of multiple by the large of and within and but is not to a to a or pairwise of the unfolding of and S-D614G showed that were at and and the between and °C showed a between the two suggesting that the stabilizing of D614G is to the of the S protein the host of analysis of and S-D614G after different of unfolding in a new this we the cryo-EM structure of S-D614G and revealed the high of conformational heterogeneity within the RBDs (Fig. to the by et L. Wang X. K.E. C. Wang Y. A. A. C. K. J.E. et and analysis of the D614G SARS-CoV-2 spike protein 2020; 183: Full Text Full Text PDF PubMed Scopus Google Scholar), we not conformational the between D614 and but we significant of the as reported by et (Fig. D.J. A.G. C. A. Xu P. S.R. The of the D614G on the structure of the spike of Natl. Acad. Sci. U. S. A. 2021; PubMed Scopus Google Scholar). The most significant is the increased resistance of S-D614G to cold- and heat-induced unfolding (Fig. the of the between D614 and is is by by the significantly total of unfolding of S-D614G with that of (Fig. We that the increased stability of S-D614G be further to the increased in the conformational heterogeneity of the RBDs (Fig. which to the of the The of findings is the increased stability the in fitness of the G which on the S protein to host receptor namely ACE2. The of the cold sensitivity of the S protein will increase the of the infection over a range of analysis of S protein at the that of the reported cryo-EM and studies of the SARS-CoV-2 S protein have by its sensitivity to cold leading to the unfolding and the of structural that to the thermal at 60 to °C (Fig. the unfolding of the S protein is to significantly the of the RBD to which ACE2 and neutralizing antibodies bind and impact of binding the ability of S-D614G to long-term at 4 °C unfolding the prefusion a to better vaccine and the to a large of mutations and (11Xiong X. Qu K. Ciazynska K.A. Hosmillo M. Carter A.P. Ebrahimi S. Ke Z. Scheres S.H.W. Bergamaschi L. Grice G.L. Zhang Y. Collaboration C.-N.C.-B. Nathan J.A. Baker S. James L.C. et al.A thermostable, closed SARS-CoV-2 spike protein trimer.Nat. Struct. Mol. Biol. 2020; 27: 934-941Crossref PubMed Scopus (162) Google Scholar, C.L. Goldsmith J.A. H.C. K. D. Lee A.G. Liu Y. Johnson J. et design of SARS-CoV-2 2020; PubMed Scopus Google Scholar, M. Walls A.C. Bowen J.E. D. Veesler D. of coronavirus spike in the closed Struct. Mol. Biol. 2020; 27: PubMed Scopus Google Scholar). The nucleotide of SARS-CoV-2 S protein a of Ma The the to of the S protein the a mutation and changes at the were for (4Wrapp D. Wang N. Corbett K.S. Goldsmith J.A. Hsieh C.L. Abiona O. Graham B.S. McLellan J.S. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation.Science. 2020; 367: 1260-1263Crossref PubMed Scopus (84) Google Scholar), which corresponds to The D614G mutation to domain on by a and a were to the of and S-D614G as T.J. Chang Y.C. P. Y.C. Chang Y.C. Wu K.H. Chang Hsu S.-T.D. Cryo-EM analysis of a coronavirus spike protein reveals a structure and Natl. Acad. Sci. U. S. A. 2020; 117: PubMed Scopus Google Scholar). 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