Structural adaptation of a thermostable biotin-binding protein in a psychrophilic environment

Shwanavidin is an avidin-like protein from the marine proteobactrium Shewanella denitrificans, which exhibits an innate dimeric structure while maintaining high affinity toward biotin. A unique residue (Phe-43) from the L3,4 loop and a distinctive disulfide bridge were shown to account for the high affinity toward biotin. Phe-43 emulates the function and position of the critical intermonomeric Trp that characterizes the tetrameric avidins but is lacking in shwanavidin. The 18 copies of the apo-monomer revealed distinctive snapshots of L3,4 and Phe-43, providing rare insight into loop flexibility, binding site accessibility, and psychrophilic adaptation. Nevertheless, as in all avidins, shwanavidin also displays high thermostability properties. The unique features of shwanavidin may provide a platform for the design of a long sought after monovalent form of avidin, which would be ideal for novel types of biotechnological application

Wilavidin* — a novel member of the avidin family that forms unique biotin‐binding hexamers

Nature's optimization of protein functions is a highly intricate evolutionary process. In addition to optimal tertiary folding, the intramolecular recognition among the monomers that generate higher-order quaternary arrangements is driven by stabilizing interactions that have a pivotal role for ideal activity. Homotetrameric avidin and streptavidin are regularly utilized in many applications, whereby their ultra-high affinity toward biotin is dependent on their quaternary arrangements. In recent years, a new subfamily of avidins was discovered that comprises homodimers rather than tetramers, in which the high affinity toward biotin is maintained. Intriguingly, several of the respective dimers have been shown to assemble into higher-order cylindrical hexamers or octamers that dissociate into dimers upon biotin binding. Here, we present wilavidin, a newly discovered member of the dimeric subfamily, forming hexamers in the apo form, which are uniquely maintained upon biotin binding with six high-affinity binding sites. Removal of the short C-terminal segment of wilavidin resulted in the presence of the dimer only, thus emphasizing the role of this segment in stabilizing the hexamer. Utilization of a hexavalent biotin-binding form of avidin would be beneficial for expanding the biotechnological toolbox. Additionally, this unique family of dimeric avidins and their propensity to oligomerize to hexamers or octamers can serve as a basis for protein oligomerization and intermonomeric recognition as well as cumulative interactions that determine molecular assemblies

Osmostress induces autophosphorylation of Hog1 via a C-terminal regulatory region that is conserved in p38α.

Many protein kinases require phosphorylation at their activation loop for induction of catalysis. Mitogen-activated protein kinases (MAPKs) are activated by a unique mode of phosphorylation, on neighboring Tyrosine and Threonine residues. Whereas many kinases obtain their activation via autophosphorylation, MAPKs are usually phosphorylated by specific, dedicated, MAPK kinases (MAP2Ks). Here we show however, that the yeast MAPK Hog1, known to be activated by the MAP2K Pbs2, is activated in pbs2Δ cells via an autophosphorylation activity that is induced by osmotic pressure. We mapped a novel domain at the Hog1 C-terminal region that inhibits this activity. Removal of this domain provides a Hog1 protein that is partially independent of MAP2K, namely, partially rescues osmostress sensitivity of pbs2Δ cells. We further mapped a short domain (7 amino acid residues long) that is critical for induction of autophosphorylation. Its removal abolishes autophosphorylation, but maintains Pbs2-mediated phosphorylation. This 7 amino acids stretch is conserved in the human p38α. Similar to the case of Hog1, it's removal from p38α abolishes p38α's autophosphorylation capability, but maintains, although reduces, its activation by MKK6. This study joins a few recent reports to suggest that, like many protein kinases, MAPKs are also regulated via induced autoactivation

Systematic mutations of the residues between F343 to Y337 do not affect Pbs2-independent phosphorylation.

<p>Phosphorylation levels of Hog1 proteins carrying the indicated mutations were tested in <i>hog1</i>Δ<i>pbs2</i>Δ cells exposed (lanes 8–14) or not exposed (lanes 1–7) to 1 M NaCl.</p

Hog1 proteins lacking the C-terminal tail are capable of partially rescuing <i>pbs2</i>Δ cells of osmostress.

<p><b>A.</b> Phosphorylation levels of wild type Hog1 (Hog1^WT) and of the truncated Hog1 proteins, Hog1^A366, Hog1^S356 and Hog1^F343 were tested when expressed in <i>hog1</i>Δ cells, before (−) and 10 minutes after (+) exposure to 1 M NaCl. <b>B.. </b><i>hog1</i>Δ cells harboring an empty vector (negative control), or vectors expressing Hog1^WT, or the truncated Hog1 proteins Hog1^A366, Hog1^S356 or Hog1^F343 were plated in five dilutions on plates containing YPD supplemented with 1 M NaCl. All strains were also plated on medium with no NaCl and all grew equally well on these plates (not shown). <b>C.. </b><i>hog1</i>Δ<i>pbs2</i>Δ cells harboring same vectors as in panel B were plated in five dilutions on plates containing YPD supplemented with 0.8 M NaCl. Growth of cells expressing an intrinsically active Hog1 variant (Hog1^D170A) served as a positive control. All strains were also plated on medium with no NaCl and all grew equally well on these plates (not shown). <b>D.</b> Phosphorylation levels of the indicated Hog1 proteins were tested as in panel A, but when expressed in <i>hog1</i>Δ<i>pbs2</i>Δ cells. Phosphorylation level of Hog1^WT, expressed in <i>hog1</i>Δ strain is presented for comparison (right lanes).</p