Biophysical insights from a single chain camelid antibody directed against the Disrupted-in-Schizophrenia 1 protein

Abstract

Accumulating evidence suggests an important role for the Disrupted-in-Schizophrenia 1 (DISC1) protein in neurodevelopment and chronic mental illness. In particular, the C-terminal 300 amino acids of DISC1 have been found to mediate important protein-protein interactions and to harbor functionally important phosphorylation sites and disease-associated polymorphisms. However, long disordered regions and oligomer-forming subdomains have so far impeded structural analysis. VHH domains derived from camelid heavy chain only antibodies are minimal antigen binding modules with appreciable solubility and stability, which makes them well suited for the stabilizing proteins prior to structural investigation. Here, we report on the generation of a VHH domain derived from an immunized Lama glama, displaying high affinity for the human DISC1 C region (aa 691–836), and its characterization by surface plasmon resonance, size exclusion chromatography and immunological techniques. The VHH-DISC1 (C region) complex was also used for structural investigation by small angle X-ray scattering analysis. In combination with molecular modeling, these data support predictions regarding the three-dimensional fold of this DISC1 segment as well as its steric arrangement in complex with our VHH antibody.

Conformational heterogeneity coupled with β-fibril formation of a scaffold protein involved in chronic mental illnesses

“…The template for the protomer of the DISC1 C-region was obtained from a model published previously, which is consistent with SAXS data [ 22 ]. The proposed β-structure was introduced into the model using MODELLER 9.23 [ 42 ] after adding restraints defining antiparallel β-strands in segments 716–737 and 739–761, respectively, via the model‐addrsr.py script.…”

“…S1 ). Given a theoretical mass of the His 6 -tagged protein of 19.4 kDa, the major species would seem to represent a monomer or a dimer (or both), but this assignment may be confounded by the elongated shape of this fragment [ 22 ]. Intriguingly, DLS measurements at 37 °C, using protein concentrations ranging from 10 to 90 µM, revealed a predominant fraction with an apparent R H of 4–4.5 nm (81.3–99.8 kDa), which may correspond to a tetramer, and another stable population at approximately 13–19 nm (1.2–2.8 MDa) that became more apparent with increasing concentration (Fig.…”

“…The human DISC1 C-region (residues 691–836) was expressed alternatively as N-terminal His 6 - or MBP-His 6 -fusion construct. The His 6 -tag construct was expressed as described previously [ 22 ] using the vector pESPRIT002 [ 20 ] for transforming E. coli BL21 (DE3) pLysE T1R cells, while the MBP-His 6 fusion construct was cloned in pET-15b and expressed in E. coli (DE3) C43 cells. The culture was grown either in Luria-Bertani (LB) broth or M9 minimal medium.…”

“…Proteins V H H B5 and NDEL1 were prepared in a similar fashion as described previously [ 22 , 27 ], which was also adopted for LIS1 expression and purification. Briefly, V H H B5, LIS1, and NDEL1 proteins were expressed in E. coli cells of the strain types Rosetta (DE3) pLysS, C43 (DE3), and BL21 (DE3) pLysE-T1R, respectively.…”

“…A systematic search for stably folded and soluble regions using the high-throughput Expression of Soluble Proteins by Random Incremental Truncation (ESPRIT) technique [ 20 ] revealed four putative domains that were mnemonically named D, I, S and C and comprise residues 257–383, 539–655, 635–738, and 691–836 [ 21 ], respectively. Among these presumably structured regions of DISC1, the C-region has been characterized most thoroughly, including its mode of binding to a camelid nanobody [ 22 ]. Solution NMR structures of a truncated version of the murine DISC1 C-region comprising the distal 73 residues have been determined in the presence of peptide fragments of ATF4 [ 23 ] and NDE1 [ 24 ], respectively.…”