Structural insights into a StART-like domain in Lam4 and its interaction with sterol ligands.

Sterols are essential components of cellular membranes and shape their biophysical properties. The recently discovered family of Lipid transfer proteins Anchored at Membrane contact sites (LAMs) has been suggested to carry out intracellular sterol traffic using StART-like domains. Here, we studied the second StART-like domain of Lam4p from S. cerevisiae by NMR. We show that NMR data are consistent with the StART-like domain structure, and that several functionally important regions within the domain exhibit significant conformational dynamics. NMR titration experiments confirm sterol binding to the canonical sterol-binding site and suggest a role of membrane interactions on the thermodynamics and kinetics of sterol binding.

Structural mass spectrometry decodes domain interaction and dynamics of the full-length Human Histone Deacetylase 2.

Human Histone Deacetylase 2 (HDAC2) belongs to a conserved enzyme superfamily that regulates deacetylation inside cells. HDAC2 is a drug target as it is known to be upregulated in cancers and neurodegenerative disorders. It consists of globular deacetylase and C-terminus intrinsically-disordered domains [1-3]. To date, there is no full-length structure of HDAC2 available due to the high intrinsic flexibility of its C-terminal domain. The intrinsically-disordered domain, however, is known to be important for the enzymatic function of HDAC2 [1, 4]. Here we combine several structural Mass Spectrometry (MS) methodologies such as denaturing, native, ion mobility and chemical crosslinking, alongside biochemical assays and molecular modelling to study the structure and dynamics of the full-length HDAC2 for the first time. We show that MS can easily dissect heterogeneity inherent within the protein sample and at the same time probe the structural arrangement of the different conformers present. Activity assays combined with data from MS and molecular modelling suggest how the structural dynamics of the C-terminal domain, and its interactions with the catalytic domain, regulate the activity of this enzyme.

Selective internuclear coupling estimation in the solid-state NMR of multiple-spin systems.

A new solid-state NMR method is presented for estimating homonuclear dipole-dipole couplings for selected groups of nuclear spins in a multiple-spin coupled network. The methodology combines off-magic-angle spinning, frequency selective spin echoes, and multiple quantum filtering. The new method is insensitive to incoherent relaxation effects and may be used to estimate weak couplings. Internuclear (13)C-(13)C couplings are estimated in uniformly (13)C-labelled l-Histidine·HCl·H(2)O. Weak intermolecular couplings between (13)C nuclei separated by distances exceeding 6 Å are estimated.

The FapF Amyloid Secretion Transporter Possesses an Atypical Asymmetric Coiled Coil.

Gram-negative bacteria possess specialized biogenesis machineries that facilitate the export of amyloid subunits, the fibers of which are key components of their biofilm matrix. The secretion of bacterial functional amyloid requires a specialized outer-membrane protein channel through which unfolded amyloid substrates are translocated. We previously reported the crystal structure of the membrane-spanning domain of the amyloid subunit transporter FapF from Pseudomonas. However, the structure of the periplasmic domain, which is essential for amyloid transport, is yet to be determined. Here, we present the crystal structure of the N-terminal periplasmic domain at 1.8-Å resolution. This domain forms a novel asymmetric trimeric coiled coil that possesses a single buried tyrosine residue as well as an extensive hydrogen-bonding network within a glutamine layer. This new structural insight allows us to understand this newly described functional amyloid secretion system in greater detail.

Supercycled homonuclear dipolar decoupling sequences in solid-state NMR.

We compare the performance of the windowed phase-modulated Lee-Goldburg (wPMLG) and the windowed decoupling using mind boggling optimisation (wDUMBO) sequences at various magic-angle spinning rates and nutation frequencies of the pulses. Additionally, we introduce a supercycled version of wDUMBO and compare its efficiency with that of the non-supercycled implementation of wDUMBO. The efficiency of the supercycled version of wPMLG, denoted wPMLG-S2, is compared with a new supercycled version of wPMLG that we notate as wPMLG-S3. The interaction between the supercycled homonuclear dipolar decoupling sequences and the sample rotation is analysed using symmetry-based selection rules.