Naima G. Sharaf

All Publications

• Expression, Purification, and Characterization of Escherichia Coli Diacylated Lipoprotein Ycjn Sharaf, N., Trevino, M. A., Amankwah, K., Fernandez, D., Weston, S., Stewart, C. J., Gallardo, J. M. WILEY. 2024: 118-119

  View details for Web of Science ID 001437110900163

• Expression, purification, and characterization of diacylated Lipo-YcjN from Escherichia coli. The Journal of biological chemistry Trevino, M. A., Amankwah, K. A., Fernandez, D., Weston, S. A., Stewart, C. J., Gallardo, J. M., Shahgholi, M., Sharaf, N. G. 2024: 107853

  Abstract

  YcjN is a putative substrate binding protein expressed from a cluster of genes involved in carbohydrate import and metabolism in Escherichia coli. Here, we determine the crystal structure of YcjN to a resolution of 1.95 A, revealing that its three-dimensional structure is similar to substrate binding proteins in subcluster D-I, which includes the well-characterized maltose binding protein (MBP). Furthermore, we found that recombinant overexpression of YcjN results in the formation of a lipidated form of YcjN that is posttranslationally diacylated at cysteine 21. Comparisons of size-exclusion chromatography profiles and dynamic light scattering measurements of lipidated and non-lipidated YcjN proteins suggest that lipidated YcjN aggregates in solution via its lipid moiety. Additionally, bioinformatic analysis indicates that YcjN-like proteins may exist in both Bacteria and Archaea, potentially in both lipidated and non-lipidated forms. Together, our results provide a better understanding of the aggregation properties of recombinantly expressed bacterial lipoproteins in solution and establish a foundation for future studies that aim to elucidate the role of these proteins in bacterial physiology.

  View details for DOI 10.1016/j.jbc.2024.107853

  View details for PubMedID 39362470

• Characterization of the ABC methionine transporter from Neisseria meningitidis reveals that lipidated MetQ is required for interaction ELIFE Sharaf, N. G., Shahgholi, M., Kim, E., Lai, J. Y., VanderVelde, D. G., Lee, A. T., Rees, D. C. 2021; 10

  Abstract

  NmMetQ is a substrate-binding protein (SBP) from Neisseria meningitidis that has been identified as a surface-exposed candidate antigen for meningococcal vaccines. However, this location for NmMetQ challenges the prevailing view that SBPs in Gram-negative bacteria are localized to the periplasmic space to promote interaction with their cognate ABC transporter embedded in the bacterial inner membrane. To elucidate the roles of NmMetQ, we characterized NmMetQ with and without its cognate ABC transporter (NmMetNI). Here, we show that NmMetQ is a lipoprotein (lipo-NmMetQ) that binds multiple methionine analogs and stimulates the ATPase activity of NmMetNI. Using single-particle electron cryo-microscopy, we determined the structures of NmMetNI in the presence and absence of lipo-NmMetQ. Based on our data, we propose that NmMetQ tethers to membranes via a lipid anchor and has dual function and localization, playing a role in NmMetNI-mediated transport at the inner membrane and moonlighting on the bacterial surface.

  View details for DOI 10.7554/eLife.69742

  View details for Web of Science ID 000693095000001

  View details for PubMedID 34409939

  View details for PubMedCentralID PMC8416018

• SARS-CoV-2 neutralizing antibody structures inform therapeutic strategies. Nature Barnes, 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, A. P., Bjorkman, P. J. 2020; 588 (7839): 682-687

  Abstract

  The coronavirus disease 2019 (COVID-19) pandemic presents an urgent health crisis. Human neutralizing antibodies that target the host ACE2 receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein1-5 show promise therapeutically and are being evaluated clinically6-8. Here, to identify the structural correlates of SARS-CoV-2 neutralization, we solved eight new structures of distinct COVID-19 human neutralizing antibodies5 in complex with the SARS-CoV-2 spike trimer or RBD. Structural comparisons allowed us to classify the antibodies into categories: (1) neutralizing antibodies encoded by the VH3-53 gene segment with short CDRH3 loops that block ACE2 and bind only to 'up' RBDs; (2) ACE2-blocking neutralizing antibodies that bind both up and 'down' RBDs and can contact adjacent RBDs; (3) neutralizing antibodies that bind outside the ACE2 site and recognize both up and down RBDs; and (4) previously described antibodies that do not block ACE2 and bind only to up RBDs9. Class 2 contained four neutralizing antibodies with epitopes that bridged RBDs, including a VH3-53 antibody that used a long CDRH3 with a hydrophobic tip to bridge between adjacent down RBDs, thereby locking the spike into a closed conformation. Epitope and paratope mapping revealed few interactions with host-derived N-glycans and minor contributions of antibody somatic hypermutations to epitope contacts. Affinity measurements and mapping of naturally occurring and in vitro-selected spike mutants in 3D provided insight into the potential for SARS-CoV-2 to escape from antibodies elicited during infection or delivered therapeutically. These classifications and structural analyses provide rules for assigning current and future human RBD-targeting antibodies into classes, evaluating avidity effects and suggesting combinations for clinical use, and provide insight into immune responses against SARS-CoV-2.

  View details for DOI 10.1038/s41586-020-2852-1

  View details for PubMedID 33045718

  View details for PubMedCentralID PMC8092461

• Structures of Human Antibodies Bound to SARS-CoV-2 Spike Reveal Common Epitopes and Recurrent Features of Antibodies. Cell Barnes, C. O., West, A. P., Huey-Tubman, K. E., Hoffmann, M. A., Sharaf, N. G., Hoffman, P. R., Koranda, N., Gristick, H. B., Gaebler, C., Muecksch, F., Lorenzi, J. C., Finkin, S., Hägglöf, T., Hurley, A., Millard, K. G., Weisblum, Y., Schmidt, F., Hatziioannou, T., Bieniasz, P. D., Caskey, M., Robbiani, D. F., Nussenzweig, M. C., Bjorkman, P. J. 2020; 182 (4): 828-842.e16

  Abstract

  Neutralizing antibody responses to coronaviruses mainly target the receptor-binding domain (RBD) of the trimeric spike. Here, we characterized polyclonal immunoglobulin Gs (IgGs) and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their focus on RBD epitopes, recognition of alpha- and beta-coronaviruses, and contributions of avidity to increased binding/neutralization of IgGs over Fabs. Using electron microscopy, we examined specificities of polyclonal plasma Fabs, revealing recognition of both S1A and RBD epitopes on SARS-CoV-2 spike. Moreover, a 3.4 Å cryo-electron microscopy (cryo-EM) structure of a neutralizing monoclonal Fab-spike complex revealed an epitope that blocks ACE2 receptor binding. Modeling based on these structures suggested different potentials for inter-spike crosslinking by IgGs on viruses, and characterized IgGs would not be affected by identified SARS-CoV-2 spike mutations. Overall, our studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.

  View details for DOI 10.1016/j.cell.2020.06.025

  View details for PubMedID 32645326

  View details for PubMedCentralID PMC7311918

• NMR structure of the HIV-1 reverse transcriptase thumb subdomain JOURNAL OF BIOMOLECULAR NMR Sharaf, N. G., Brereton, A. E., Byeon, I. L., Karplus, P., Gronenborn, A. M. 2016; 66 (4): 273-280

  Abstract

  The solution NMR structure of the isolated thumb subdomain of HIV-1 reverse transcriptase (RT) has been determined. A detailed comparison of the current structure with dozens of the highest resolution crystal structures of this domain in the context of the full-length enzyme reveals that the overall structures are very similar, with only two regions exhibiting local conformational differences. The C-terminal capping pattern of the αH helix is subtly different, and the loop connecting the αI and αJ helices in the p51 chain of the full-length p51/p66 heterodimeric RT differs from our NMR structure due to unique packing interactions in mature RT. Overall, our data show that the thumb subdomain folds independently and essentially the same in isolation as in its natural structural context.

  View details for DOI 10.1007/s10858-016-0077-2

  View details for Web of Science ID 000392076700006

  View details for PubMedID 27858311

  View details for PubMedCentralID PMC5218889