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The following paper (PDF attached) describes why the above sequence might be important:
https://science.sciencemag.org/content/early/2020/04/02/science.abb7269
According to the paper:
Protein Engineering, Spring 2020Prof. T. Cardozo FINAL EXAM The following is an amino acid sequence of a single protein domain/fold/topology: QMQLVQSGTEVKKPGESLKISCKGSGFGFITLWIGWVRQMPGKGLEWMGIIYPGDSETRYSPSFQGQVTISADKSINTAYLQWSSLKASDTAIYYCAGGSGISTPMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS The following paper (PDF attached) describes why the above sequence might be important: https://science.sciencemag.org/content/early/2020/04/02/science.abb7269 According to the paper: 1. (8 points) The above sequence is involved in which human disease? 2. (8 points) The epitope targeted by the CR3022 antibody may not be immunogenic because: a. The epitope is not large enough b. The epitope is not exposed on the protein surface c. The epitope is not the right shape d. The epitope is not complex enough 3. (8 points) Is the above sequence part of an antigen or an antibody? 4. (12 points) The above sequence is different from any 3D structure in the Protein Data Bank. It is, however, very similar to VH (heavy chain variable) domain in the CR3022 monoclonal antibody, which can serve as a template for modeling it (attached). Submit an ICM icb file containing ONLY a 3D structural model of the above sequence. 5. Answer the following a. To predict the structure of the area that is different between the model and the template, is loop modeling or side chain modeling required? Why? (8 points) b. To predict the structure of the area that is different between the model and the template, which conformational search strategy should be used? (8 points) c. To predict the structure of the area that is different between the model and the template, what energy terms should be used? (8 points) d. How does the optimal docking area (ODA) change between the model and the template? Submit a side-by-side picture of the two for comparison (8 points) e. How does the electrostatic surface change between the model and the template? Submit a side-by-side picture of the two for comparison (8 points) 6. The paper describes the CR0233 monoclonal antibody. The following questions are about that antibody (8 points each) a. Is it human, humanized, non-human or chimeric? b. How would it be delivered to a person as a drug treatment: pill, injection or cream? 7. (8 points) How might the CR3022 monoclonal cited in the paper be improved by protein engineering? a. Redesign a CR0233 surface in ICM to bind to a different protein b. Redesign a CR0233 surface in ICM to be more immunogenic c. Redesign the CR0233 CDRH2 loop to bind better to a glycan d. Use phage display to screen for CR3022 mutants that bind better to the SARS-CoV-2 spike protein Submit both the written answers to questions and the ICM.icb Project file to me via NYU Classes no later than the deadline stated in the class syllabus. A highly conserved cryptic epitope in the receptor-binding domains of SARS-CoV-2 and SARS-CoV Cite as: M. Yuan et al., Science 10.1126/science.abb7269 (2020). REPORTS First release: 3 April 2020 www.sciencemag.org (Page numbers not final at time of first release) 1 The ongoing outbreak of Coronavirus Disease 2019 (COVID- 19) originally emerged in China during December 2019 (1) and had become a global pandemic by March 2020. COVID- 19 is caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (2). Two other corona- viruses have caused world-wide outbreaks in the past two decades, namely SARS-CoV (2002–2003) and Middle East res- piratory syndrome coronavirus (MERS-CoV) (2012–present). The surface spike glycoprotein (S), which is critical for virus entry through engaging the host receptor and mediating vi- rus-host membrane fusion, is the major antigen of corona- viruses. The S proteins of SARS-CoV-2 and SARS-CoV, which are phylogenetically closely related, have an amino-acid se- quence identity of around 77% (3). Such a high degree of se- quence similarity raises the possibility that cross-reactive epitopes may exist. CR3022, which was previously isolated from a convales- cent SARS patient, is a neutralizing antibody that targets the receptor-binding domain (RBD) of SARS-CoV (4). The immu- noglobulin heavy chain variable, diversity, and joining (IGHV, IGHD, and IGHJ) regions are encoded by germline genes IGHV5-51, IGHD3-10, and IGHJ6, while the light chain variable and joining regions are encoded by IGKV4-1 and IGKJ2 (4). Based on IgBlast analysis (5), the IGHV of CR3022 is 3.1% somatically mutated at the nucleotide sequence level, which results in eight amino-acid changes from the germline sequence, whereas IGKV of CR3022 is 1.3% somatically mu- tated resulting in three amino-acid changes from the germline sequence (fig. S1). A recent study has shown that CR3022 can also bind to the RBD of SARS-CoV-2 (6). This finding provides an opportunity to uncover a cross-reactive epitope. We therefore determined the crystal structure of CR3022 with the SARS-CoV-2 RBD (Fig. 1A) at 3.1 Å resolu- tion (table S1 and fig. S2, A and B) (7). CR3022 uses both heavy and light chains (Fig. 1B), and all six complementarity- determining region (CDR) loops (Fig. 1C) for interaction with the RBD. The buried surface area on the epitope is 917 Å2 and SARS-CoV-2 recognition by CR3022 is largely driven by hy- drophobic interactions (Fig. 1E). Five out of 11 somatic muta- tions are found in the paratope region (defined as residues on the antibody buried by RBD) (fig. S2C), implying their likely importance in the affinity maturation process. Out of 28 residues in the epitope (defined as residues bur- ied by CR3022), 24 (86%) are conserved between SARS-CoV- 2 and SARS-CoV (Figs. 1D and 2A). This high sequence con- servation explains the cross-reactivity of CR3022. Nonethe- less, despite having a high conservation of the epitope residues, CR3022 Fab binds to SARS-CoV RBD (Kd = 1 nM) with a much higher affinity than to SARS-CoV-2 RBD (Kd = 115 nM) (Table 1 and fig. S3). The difference in binding affin- ity of CR3022 between SARS-CoV-2 and SARS-CoV RBDs is likely due to the non-conserved residues in the epitope (Fig. 2). The most dramatic difference is an additional N-glycosyl- ation site at N370 on SARS-CoV (N357 in SARS-CoV number- ing). The N-glycan sequon (NxS/T) arises from an amino-acid difference at residue 372, where SARS-CoV has a Thr com- pared to Ala in SARS-CoV-2 (Fig. 2B). Mass spectrometry analysis shows that a complex glycan is indeed present at this A highly conserved cryptic epitope in the receptor-binding domains of SARS-CoV-2 and SARS-CoV Meng Yuan1*, Nicholas C. Wu1*, Xueyong Zhu1, Chang-Chun D. Lee1, Ray T. Y. So2, Huibin Lv2, Chris K. P. Mok2†, Ian A. Wilson1,3† 1Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. 2HKU-Pasteur Research Pole, School of Public Health, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China. 3The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, 92037, USA. *These authors contributed equally to this work. †Corresponding author. Email:
[email protected] (C.K.P.M.);
[email protected] (I.A.W.) The outbreak of COVID-19 caused by SARS-CoV-2 virus has now become a pandemic, but there is currently very little understanding of the antigenicity of the virus. We therefore determined the crystal structure of CR3022, a neutralizing antibody previously isolated from a convalescent SARS patient, in complex with the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein to 3.1 Å. CR3022 targets a highly conserved epitope, distal from the receptor-binding site, that enables cross-reactive binding between SARS-CoV-2 and SARS-CoV. Structural modeling further demonstrates that the binding epitope can only be accessed by CR3022 when at least two RBD on the trimeric S protein are in the “up” conformation and slightly rotated. Overall, this study provides molecular insights into antibody recognition of SARS-CoV-2. on A pril 7, 2020 http://science.sciencem ag.org/ D ow nloaded from http://www.sciencemag.org/ http://science.sciencemag.org/ First release: 3 April 2020 www.sciencemag.org (Page numbers not final at time of first release) 2 N-glycosylation site in SARS-CoV (8). An N-glycan at N370 would fit into a groove formed between heavy and light chains (Fig. 2C), which could increase contact and, hence, binding affinity to CR3022. This result also suggests that the difference in antigenicity between the RBDs of SARS-CoV-2 and SARS-CoV can at least be partially attributed to the N- glycosylation site at residue 370. We tested whether CR3022 was able to neutralize SARS-CoV-2 and SARS-CoV in an in vitro microneutralization assay (7). While CR3022 could neu- tralize SARS-CoV, it did not neutralize SARS-CoV-2 at the highest concentration tested (400 μg/mL) (fig. S4). This in vitro neutralization result is consistent with lower affinity binding of CR3022 for SARS-CoV-2, although other explana- tions are possible as outlined below. SARS-CoV-2 uses the same host receptor, angiotensin I converting enzyme 2 (ACE2) as SARS-CoV (3, 9–11). Interest- ingly, the epitope of CR3022 does not overlap with the ACE2- binding site (Fig. 3A). Structural alignment of the CR3022- SARS-CoV-2 RBD complex with the ACE2-SARS-CoV-2 RBD complex (11) further indicates that binding of CR3022 would not clash with ACE2 (12). This analysis implies that the neu- tralization mechanism of CR3022 for SARS-CoV does not de- pend on direct blocking of receptor binding, which is consistent with the observation that CR3022 does not com- pete with ACE2 for binding to the RBD (6). Unlike CR3022, most known SARS RBD-targeted antibodies compete with ACE2 for binding to RBD (4, 13–16). The epitopes of these an- tibodies are very different from that of CR3022 (Fig. 3B). In fact, it has been shown that CR3022 can synergize with other RBD-targeted antibodies to neutralize SARS-CoV (4). Alt- hough CR3022 itself cannot neutralize SARS-CoV-2 in this in vitro assay, whether CR3022 can synergize with other SARS- CoV-2 RBD-targeted monoclonal antibodies for neutraliza- tion remains to be determined. The recently determined cryo-EM structure of homotri- meric SARS-CoV-2 S protein (17, 18) demonstrated that the RBD, as in other coronaviruses (19, 20) can undergo a hinge- like movement to transition between “up” or “down” confor- mations (Fig. 4A). ACE2 host receptor can only interact with the RBD when it is in the “up” conformation, whereas the “down” conformation is inaccessible to ACE2. Interestingly, the epitope of CR3022 is also only accessible when the RBD is in the “up” conformation (Fig. 4, B and C). However, even