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US-12624092-B2 - Compositions and methods for membrane protein structure determination

US12624092B2US 12624092 B2US12624092 B2US 12624092B2US-12624092-B2

Abstract

Disclosed herein are compositions and methods for determining the structure of a membrane protein. An epitope from a membrane-proximal external region (MPER) from a viral envelope protein can be grafted on to a variety of diverse membrane proteins to allow for binding structurally characterized antibody fragments, which can aid structural studies.

Inventors

  • Benjamin C. McIlwain
  • Randy B. Stockbridge

Assignees

  • THE REGENTS OF THE UNIVERSITY OF MICHIGAN

Dates

Publication Date
20260512
Application Date
20210506

Claims (10)

  1. 1 . A complex comprising: (a) a MPER peptide fusion protein comprising formula A-B, wherein: A is a peptide having consisting of the sequence of SEQ ID NO: 1; and B is membrane protein; and (b) an antibody fragment that binds to the peptide A.
  2. 2 . The complex of claim 1 , wherein B is a membrane protein having a molecular weight of less than 200 kDa.
  3. 3 . The complex of claim 1 , wherein A is attached to the N-terminus of the membrane protein B.
  4. 4 . The complex of claim 1 , wherein the antibody fragment is a Fab fragment from an antibody selected from the group consisting of 10E8, LN01, DH511, VRC42, PGZL1, and 4E10.
  5. 5 . The complex of claim 4 , wherein the antibody fragment is a Fab fragment from an antibody selected from the group consisting of 10E8 and VRC42.
  6. 6 . A method of determining the structure of a membrane protein, comprising: (a) providing a complex of claim 1 ; and (b) determining the structure of the complex by X-ray crystallography or electron microscopy, to thereby determine the structure of the membrane protein.
  7. 7 . The method of claim 6 , wherein the structure of the complex is determined by X-ray crystallography.
  8. 8 . The method of claim 7 , further comprising a step of forming a crystal of the complex.
  9. 9 . The method of claim 6 , wherein the structure of the complex is determined by electron microscopy.
  10. 10 . The method of claim 9 , wherein the electron microscopy is cryo-electron microscopy.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a U.S. national stage filing of International Patent Application No. PCT/US2021/031093, filed on May 6, 2021, which claims priority to U.S. Provisional Patent Application No. 63/020,769, filed on May 6, 2020, the entire contents of each of which are fully incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under GM128768 awarded by the National Institutes of Health. The government has certain rights in the invention. SEQUENCE LISTING The text of the computer readable sequence listing filed herewith, titled “38317-601_Sequence_Listing_ST25”, created May 6, 2021, having a file size of 16,274 bytes, is hereby incorporated by reference in its entirety. BACKGROUND Limited tools exist for the structural analysis of small membrane proteins (e.g., those less than 200 kDa or 100 kDa). A biochemically tractable protein might not crystallize due to lack of lattice-forming crystal contacts. At the same time, such proteins are too small and indistinct to be visualized with electron microscopy (EM) and often suffer from low signal-to-noise-induced misalignment in a disordered detergent micelle (Herzik et al. Nat. Commun. 10, 1-9 (2019)). One strategy to overcome these challenges is to use soluble chaperone proteins such as antibody fragments. Two such fragments are the single-chain variable-domain fragment (scFv) and fragment antigen-binding (Fab). scFv fragments are composed of a single 25-kDa unit, the variable domain of an antibody joined by a linker; scFvs are often extremely rigid, leading to highly ordered crystals. Fabs have been used in membrane protein structural biology, primarily in X-ray crystallography. Antibody fragments also been used in high resolution electron microscopy (Wu et al. Structure 20, 582-592 (2012)). Fab fragments have two 25-kDa units, the constant and variable domains, which are arranged as an open clamshell through two elbow regions. The elbow-like hinge region between these units appears as a hole—a feature particularly useful for high-resolution particle alignment from EM images of particles in either vitrified ice or negative stain. Additionally, the 50-kDa proteins are an excellent strategy to increase the effective size of complexed particles, and can overcome problems with preferred particle orientation, reducing anisotropy of the dataset by improving the distribution of Euler angles of the particles in single particle cryo-EM analysis (Dang et al. Nature, 552, 426-429 (2017); Butterwick et al. Nature 560,447-485 (2018)). Antibody fragments that bind targets specifically can also be used as localization tags, which are useful for interpreting low-resolution EM density maps in order to unambiguously localize regions of the protein and map macromolecule topology. However, several non-trivial limitations accompany the use of antibody fragments for structural biology. Antibodies with binding specificities to a target protein are generally discovered by immunization of the target protein in small laboratory animals. The requisite immunization and antibody-discovery campaign can take several months, and it can be difficult to generate antibodies against small membrane proteins, which can be poorly immunogenic. Antibody fragments discovered by this method sometimes lack stability or biochemical tractability, and flexible loops with limited utility for structural studies are often recognized. Additional complications arise if antibodies are desired against a structural target in a particular confirmation or a substrate-occupied state. The development of combinatorial libraries of antibody-like proteins, such as megabodies, nanobodies, and monobodies, has addressed some of these problems, allowing binder discovery via phage or yeast display (Sha et al. Protein Sci., 26 (2017), pp. 910-924; Uchanski et al. Nat. Methods, 18 (2021), pp. 60-68; McMahon et al. Nat. Struct. Mol. Biol., 25 (2018), pp. 289-296). However, these approaches still require a discovery campaign and tailored approaches to select binders against a desired epitope. Identification of “plug-and-play” chaperones or fiducial markers that can be used for many different protein targets has been a recent focus of protein engineering (Kim et al. Proc. Natl. Acad. Sci., 116 (2019), pp. 17786-17791; Dutka et al. Structure, 27 (1862-74) (2019), Article e7; Yeates et al. Curr. Opin. Struct. Biol., 60 (2020), pp. 142-149; Mukherjee et al. Nat. Commun., 11 (2020), p. 1598). In particular, anti-helix antibodies that recognize a short, linear epitope with α-helical secondary structure have been put forth as a promising avenue for the development of unobtrusive, broadly applicable, high-affinity Fab recognition (Kim 2019; Koide Proc. Natl. Acad. Sci. U.S.A, 116 (2019), pp. 17611-17613). Such an approach has special potential for determining the structures of small membrane proteins. A general drawback of t