Protein in-cell NMR in Escherichia coli

The inside of the cell is a crowded and complex environment that is impossible to duplicate by studying proteins and other molecules in dilute solution. The effects of macromolecular crowding are potentially important to all cellular functions, but until recently studies have been conducted mostly in dilute solution. In-cell Nuclear Magnetic Resonance (NMR) spectroscopy is becoming an important tool in studying proteins under physiologically relevant conditions. In some instances, however, protein signals from leaked protein are seen in the supernatant surrounding the in-cell slurry. I examined how expression levels contribute to protein leakage. I also describe a device and system that provides a controlled environment for NMR experiments in living cells. I have utilized this device to study the expression of the natively disordered protein - synuclein, inside Escherichia coli. In the future, we hope to make progress in using this device to study proteins in eukaryotic cells with NMR

A Co-receptor-mimicking Antibody Stabilizes the Displaced V1V2 Loops in a Partially Open sCD4-bound HIV-1 Envelope Complex

Background: HIV-1 envelope glycoprotein (Env), a trimer of gp120-gp41 heterodimers, mediates membrane fusion after binding host receptor CD4. Receptor binding to closed Env displaces the V1V2 loops from Env‘s apex, allowing co-receptor binding and opening Env to enable gp41-mediated fusion. Methods: Using single-particle cryoEM methods, we solved the sCD4-bound structure of a clade B B41 SOSIP trimer in complex with 21c, a CD4-induced (CD4i) coreceptor-mimicking antibody, and with 8ANC195, a broadly neutralizing antibody (bNAb) that recognizes the gp120-gp41 interface, to a resolution of 4.05 Å. Results: Despite binding of the 8ANC195 Fab that partially closes the open, sCD4-bound Env conformation, our structure shows rearrangements in gp120, including displacement of V1V2, exposure of V3, formation of the 4-stranded bridging sheet, and formation of the α0 helix. In addition, unlike the V1V2 regions in similar sCD4-bound Env structures complexed with the CD4i antibody 17b, the displaced V1V2 loops in the B41-sCD4-21c-8ANC195 structure exhibited ordered density allowing the structure of the displaced V1V2 to be determined for the first time. Comparing partially- and fully-open Envs with closed Envs shows that gp41 rearrangements are independent of CD4-induced gp120 bridging sheet formation and V1V2 displacement, suggesting an order of conformational changes before co-receptor binding: (i) gp120 opening inducing side chain rearrangements and a compact gp41 central helix conformation, and (ii) bridging sheet formation, and V1V2 displacement. Conclusions: Analyses of these results further our understanding of HIV-1 Env conformational changes leading to fusion and provide templates for designing agents to disrupt HIV-1 entry into target cells

In-cell protein NMR and protein leakage

In-cell nuclear magnetic resonance spectroscopy is a tool for studying proteins under physiologically relevant conditions. In some instances, however, protein signals from leaked protein are observed in the liquid surrounding the cells. Here, we examine the expression of four proteins in Escherichia coli. We describe the controls that should be used for in-cell NMR experiments, and show that leakage is likely when the protein being studied exceeds approximately 20% of the total cellular protein

Partially Open HIV-1 Envelope Structures Exhibit Conformational Changes Relevant for Coreceptor Binding and Fusion

HIV-1 Env, a trimer of gp120-gp41 heterodimers, mediates membrane fusion after binding host receptor CD4. Receptor binding displaces V1V2 loops from Env's apex, allowing coreceptor binding and opening Env to enable gp41-mediated fusion. We present 3.54 Å and 4.06 Å cryoelectron microscopy structures of partially open soluble native-like Env trimers (SOSIPs) bound to CD4. One structure, a complex with a coreceptor-mimicking antibody that binds both CD4 and gp120, stabilizes the displaced V1V2 and reveals its structure. Comparing partially and fully open Envs with closed Envs shows that gp41 rearrangements are independent of the CD4-induced rearrangements that result in V1V2 displacement and formation of a 4-stranded bridging sheet. These findings suggest ordered conformational changes before coreceptor binding: (1) gp120 opening inducing side-chain rearrangements and a compact gp41 central helix conformation, and (2) 4-stranded bridging-sheet formation and V1V2 displacement. These analyses illuminate potential receptor-induced Env changes and inform design of therapeutics disrupting viral entry

Cryo-EM Structures of HIV-1 trimer bound to CD4-mimetics M48U1 and BNM-III-170 adopt a CD4-bound open conformation

Human Immunodeficiency Virus-1 (HIV-1), the causative agent of AIDS, impacts millions of people. Entry into target cells is mediated by the HIV-1 envelope (Env) glycoprotein interacting with host receptor CD4, which triggers conformational changes allowing binding to a coreceptor and subsequent membrane fusion. Small molecule or peptide CD4-mimetic drugs mimic CD4’s Phe43 interaction with Env by inserting into the conserved Phe43 pocket on Env subunit gp120. Here, we present single-particle cryo-EM structures of CD4-mimetics BNM-III-170 and M48U1 bound to a BG505 native-like Env trimer plus the CD4-induced antibody 17b at 3.7Å and 3.9Å resolution, respectively. CD4-mimetic-bound BG505 exhibits canonical CD4-induced conformational changes including trimer opening, formation of the 4-stranded gp120 bridging sheet, displacement of the V1V2 loop, and formation of a compact and elongated gp41 HR1C helical bundle. We conclude that CD4-induced structural changes on both gp120 and gp41 Env subunits are induced by binding to the gp120 Phe43 pocket

A Co-receptor-mimicking Antibody Stabilizes the Displaced V1V2 Loops in a Partially Open sCD4-bound HIV-1 Envelope Complex

Background: HIV-1 envelope glycoprotein (Env), a trimer of gp120-gp41 heterodimers, mediates membrane fusion after binding host receptor CD4. Receptor binding to closed Env displaces the V1V2 loops from Env‘s apex, allowing co-receptor binding and opening Env to enable gp41-mediated fusion. Methods: Using single-particle cryoEM methods, we solved the sCD4-bound structure of a clade B B41 SOSIP trimer in complex with 21c, a CD4-induced (CD4i) coreceptor-mimicking antibody, and with 8ANC195, a broadly neutralizing antibody (bNAb) that recognizes the gp120-gp41 interface, to a resolution of 4.05 Å. Results: Despite binding of the 8ANC195 Fab that partially closes the open, sCD4-bound Env conformation, our structure shows rearrangements in gp120, including displacement of V1V2, exposure of V3, formation of the 4-stranded bridging sheet, and formation of the α0 helix. In addition, unlike the V1V2 regions in similar sCD4-bound Env structures complexed with the CD4i antibody 17b, the displaced V1V2 loops in the B41-sCD4-21c-8ANC195 structure exhibited ordered density allowing the structure of the displaced V1V2 to be determined for the first time. Comparing partially- and fully-open Envs with closed Envs shows that gp41 rearrangements are independent of CD4-induced gp120 bridging sheet formation and V1V2 displacement, suggesting an order of conformational changes before co-receptor binding: (i) gp120 opening inducing side chain rearrangements and a compact gp41 central helix conformation, and (ii) bridging sheet formation, and V1V2 displacement. Conclusions: Analyses of these results further our understanding of HIV-1 Env conformational changes leading to fusion and provide templates for designing agents to disrupt HIV-1 entry into target cells