Transfer of a Rational Crystal Contact Engineering Strategy between Diverse Alcohol Dehydrogenases

Protein crystallization can serve as a purification step in biotechnological processes but is often limited by the non-crystallizability of proteins. Enabling or improving crystallization is mostly achieved by high-throughput screening of crystallization conditions and, more recently, by rational crystal contact engineering. Two selected rational crystal contact mutations, Q126K and T102E, were transferred from the alcohol dehydrogenases of Lactobacillus brevis (LbADH) to Lactobacillus kefir (LkADH). Proteins were expressed in E. coli and batch protein crystallization was performed in stirred crystallizers. Highly similar crystal packing of LkADH wild type compared to LbADH, which is necessary for the transfer of crystal contact engineering strategies, was achieved by aligning purification tag and crystallization conditions, as shown by X-ray diffraction. After comparing the crystal sizes after crystallization of LkADH mutants with the wild type, the mean protein crystal size of LkADH mutants was reduced by 40–70% in length with a concomitant increase in the total amount of crystals (higher number of nucleation events). Applying this measure to the LkADH variants studied results in an order of crystallizability T102E > Q126K > LkADH wild type, which corresponds to the results with LbADH mutants and shows, for the first time, the successful transfer of crystal contact engineering strategies.

A new crystal form of GABARAPL2

The Atg8 protein family comprises the GABA type A receptor-associated proteins (GABARAPs) and microtubule-associated protein 1 light chains 3 (MAP1LC3s) that are essential mediators of autophagy. The LC3-interacting region (LIR) motifs of autophagy receptors and adaptors bind Atg8 proteins to promote autophagosome formation, cargo recruitment, and autophagosome closure and fusion to lysosomes. A crystal structure of human GABARAPL2 has been published [PDB entry 4co7; Ma et al. (2015), Biochemistry, 54, 5469–5479]. This was crystallized in space group P21 with a monoclinic angle of 90° and shows a pseudomerohedral twinning pathology. This article reports a new, untwinned GABARAPL2 crystal form, also in space group P21, but with a 98° monoclinic angle. No major conformational differences were observed between the structures. In the structure described here, the C-terminal Phe117 binds into the LIR docking site (LDS) of a neighbouring molecule within the asymmetric unit, as observed in the previously reported structure. This crystal contact blocks the LDS for co-crystallization with ligands. Phe117 of GABARAPL2 is normally removed during biological processing by Atg4 family proteases. These data indicate that to establish interactions with the LIR, Phe117 should be removed to eliminate the crystal contact and liberate the LDS for co-crystallization with LIR peptides.

Chimeric single α-helical domains as rigid fusion protein connections for protein nanotechnology and structural biology

SummaryChimeric fusion proteins are essential tools for protein nanotechnology. Non-optimized protein-protein connections are usually flexible, which makes them unsuitable as structural building blocks. Here we show that the ER/K motif, a single α-helical domain (SAH)1, can be seamlessly fused2 to terminal helices of proteins, forming an extended and partially free-standing rigid helix. Through the intrinsic stability of the SAH, two domains can be connected with a defined distance and orientation. We designed three constructs termed YFPnano, T4Lnano, and MoStoNano, and we show that a single SAH allows the connection of two separate structural domains with sufficient rigidity to form ordered crystals. The analysis of experimentally determined structures and molecular dynamics simulations reveals a certain degree of plasticity in the connections that allows the adaptation to crystal contact opportunities. Our data show that SAHs can be stably integrated into designed structural elements, enabling new possibilities for protein nanotechnology, for example to improve the exposure of epitopes on nanoparticles (structural vaccinology), to engineer crystal contacts with minimal impact in construct flexibility (for the study of protein dynamics), and to design novel biomaterials.

Structure of apo flavin-dependent halogenase Xcc4156 hints at a reason for cofactor-soaking difficulties

Flavin-dependent halogenases regioselectively introduce halide substituents into electron-rich substrates under mild reaction conditions. For the enzyme Xcc4156 from Xanthomonas campestris, the structure of a complex with the cofactor flavin adenine dinucleotide (FAD) and a bromide ion would be of particular interest as this enzyme exclusively brominates model substrates in vitro. Apo Xcc4156 crystals diffracted to 1.6 Å resolution. The structure revealed an open substrate-binding site lacking the loop regions that close off the active site and contribute to substrate binding in tryptophan halogenases. Therefore, Xcc4156 might accept larger substrates, possibly even peptides. Soaking of apo Xcc4156 crystals with FAD led to crumbling of the intergrown crystals. Around half of the crystals soaked with FAD did not diffract, while in the others there was no electron density for FAD. The FAD-binding loop, which changes its conformation between the apo and the FAD-bound form in related enzymes, is involved in a crystal contact in the apo Xcc4156 crystals. The conformational change that is predicted to occur upon FAD binding would disrupt this crystal contact, providing a likely explanation for the destruction of the apo crystals in the presence of FAD. Soaking with only bromide did not result in bromide bound to the catalytic halide-binding site. Simultaneous soaking with FAD and bromide damaged the crystals more severely than soaking with only FAD. Together, these latter two observations suggest that FAD and bromide bind to Xcc4156 with positive cooperativity. Thus, apo Xcc4156 crystals provide functional insight into FAD and bromide binding, even though neither the cofactor nor the halide is visible in the structure.

The phospholamban pentamer interacts with the sarcoplasmic reticulum calcium pump SERCA

ABSTRACTThe interaction of phospholamban with the sarcoplasmic reticulum calcium pump (SERCA) is a major regulatory axis in cardiac muscle contractility. The prevailing model involves reversible inhibition of SERCA by monomeric phospholamban and storage of phospholamban as an inactive pentamer. However, this paradigm has been challenged by studies demonstrating that phospholamban remains associated with SERCA and that the phospholamban pentamer is required for cardiac contractility. We have previously used two-dimensional crystallization and electron microscopy to study the interaction between SERCA and phospholamban. To further understand this interaction, we compared small helical crystals and large two-dimensional crystals of SERCA in the absence and presence of phospholamban. In both crystal forms, SERCA molecules are organized into identical anti-parallel dimer ribbons. The dimer ribbons pack together with distinct crystal contacts in the helical versus large two-dimensional crystals, which allow phospholamban differential access to potential sites of interaction with SERCA. Nonetheless, we show that a phospholamban oligomer interacts with SERCA in a similar manner in both crystal forms. In the two-dimensional crystals, a phospholamban pentamer interacts with transmembrane segments M3 of SERCA and participates in a crystal contact that bridges neighboring SERCA dimer ribbons. In the helical crystals, an oligomeric form of phospholamban also interacts with M3 of SERCA, though the phospholamban oligomer straddles a SERCA-SERCA crystal contact. We conclude that the pentameric form of phospholamban interacts with SERCA, and that it plays distinct structural and functional roles in SERCA regulation.