DYNLL2 dynein light chain binds to an extended linear motif of myosin 5a tail that has structural plasticity

LC8 dynein light chains (DYNLL) are conserved homodimeric eukaryotic hub proteins that participate in diverse cellular processes. Among the binding partners of DYNLL2, myosin 5a (myo5a) is a motor protein involved in cargo transport. Here we provide a profound characterization of the DYNLL2 binding motif of myo5a in free and DYNLL2 bound form by using NMR spectroscopy, X-ray crystallography and molecular dynamics simulations. In the free form the DYNLL2 binding region, located in an intrinsically disordered domain of the myo5a tail, has a nascent helical character. The motif becomes structured and folds into a β-strand upon binding to DYNLL2. Despite all differences of the myo5a sequence from the consensus binding motif, it accommodates into the same DYNLL2 binding groove as all other partners do. Interestingly, while the core motif shows similar interaction pattern in the binding groove as seen in other complexes, the flanking residues make several additional contacts, thereby lengthening the binding motif. The N-terminal extension folds back and partially blocks the free edge of the β-sheet formed by the binding motif itself. The C-terminal extension contacts the dimer interface and interacts with symmetry related residues of the second myo5a peptide. The involvement of flanking residues of the core binding site of myo5a could modify the quaternary structure of the full-length myo5a and affect its biological functions. The presented structural data widen our understanding of the diverse partner recognition of DYNLL proteins and provide an example of a Janus-faced linear motif.

The Sodium Sialic Acid Symporter From Staphylococcus aureus Has Altered Substrate Specificity

Mammalian cell surfaces are decorated with complex glycoconjugates that terminate with negatively charged sialic acids. Commensal and pathogenic bacteria can use host-derived sialic acids for a competitive advantage, but require a functional sialic acid transporter to import the sugar into the cell. This work investigates the sodium sialic acid symporter (SiaT) from Staphylococcus aureus (SaSiaT). We demonstrate that SaSiaT rescues an Escherichia coli strain lacking its endogenous sialic acid transporter when grown on the sialic acids N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc). We then develop an expression, purification and detergent solubilization system for SaSiaT and demonstrate that the protein is largely monodisperse in solution with a stable monomeric oligomeric state. Binding studies reveal that SaSiaT has a higher affinity for Neu5Gc over Neu5Ac, which was unexpected and is not seen in another SiaT homolog. We develop a homology model and use comparative sequence analyses to identify substitutions in the substrate-binding site of SaSiaT that may explain the altered specificity. SaSiaT is shown to be electrogenic, and transport is dependent upon more than one Na+ ion for every sialic acid molecule. A functional sialic acid transporter is essential for the uptake and utilization of sialic acid in a range of pathogenic bacteria, and developing new inhibitors that target these transporters is a valid mechanism for inhibiting bacterial growth. By demonstrating a route to functional recombinant SaSiaT, and developing the in vivo and in vitro assay systems, our work underpins the design of inhibitors to this transporter

Directed evolution reveals the binding motif preference of the LC8/DYNLL hub protein and predicts large numbers of novel binders in the human proteome.

LC8 dynein light chain (DYNLL) is a eukaryotic hub protein that is thought to function as a dimerization engine. Its interacting partners are involved in a wide range of cellular functions. In its dozens of hitherto identified binding partners DYNLL binds to a linear peptide segment. The known segments define a loosely characterized binding motif: [D/S](-4)K(-3)X(-2)[T/V/I](-1)Q(0)[T/V](1)[D/E](2). The motifs are localized in disordered segments of the DYNLL-binding proteins and are often flanked by coiled coil or other potential dimerization domains. Based on a directed evolution approach, here we provide the first quantitative characterization of the binding preference of the DYNLL binding site. We displayed on M13 phage a naïve peptide library with seven fully randomized positions around a fixed, naturally conserved glutamine. The peptides were presented in a bivalent manner fused to a leucine zipper mimicking the natural dimer to dimer binding stoichiometry of DYNLL-partner complexes. The phage-selected consensus sequence V(-5)S(-4)R(-3)G(-2)T(-1)Q(0)T(1)E(2) resembles the natural one, but is extended by an additional N-terminal valine, which increases the affinity of the monomeric peptide twentyfold. Leu-zipper dimerization increases the affinity into the subnanomolar range. By comparing crystal structures of an SRGTQTE-DYNLL and a dimeric VSRGTQTE-DYNLL complex we find that the affinity enhancing valine is accommodated in a binding pocket on DYNLL. Based on the in vitro evolved sequence pattern we predict a large number of novel DYNLL binding partners in the human proteome. Among these EML3, a microtubule-binding protein involved in mitosis contains an exact match of the phage-evolved consensus and binds to DYNLL with nanomolar affinity. These results significantly widen the scope of the human interactome around DYNLL and will certainly shed more light on the biological functions and organizing role of DYNLL in the human and other eukaryotic interactomes

Terahertz absorption of illuminated photosynthetic reaction center solution: A signature of photoactivation?

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Structural Characterization of Bacterioferritin from <em>Blastochloris viridis</em>

<div><p>Iron storage and elimination of toxic ferrous iron are the responsibility of bacterioferritins in bacterial species. Bacterioferritins are capable of oxidizing iron using molecular oxygen and import iron ions into the large central cavity of the protein, where they are stored in a mineralized form. We isolated, crystallized bacterioferritin from the microaerophilic/anaerobic, purple non-sulfur bacterium <em>Blastochloris viridis</em> and determined its amino acid sequence and X-ray structure. The structure and sequence revealed similarity to other purple bacterial species with substantial differences in the pore regions. Static 3- and 4-fold pores do not allow the passage of iron ions even though structural dynamics may assist the iron gating. On the other hand the B-pore is open to water and larger ions in its native state. In order to study the mechanism of iron import, multiple soaking experiments were performed. Upon Fe(II) and urea treatment the ferroxidase site undergoes reorganization as seen in bacterioferritin from <em>Escherichia coli</em> and <em>Pseudomonas aeruginosa</em>. When soaking with Fe(II) only, a closely bound small molecular ligand is observed close to Fe₁ and the coordination of Glu94 to Fe₂ changes from bidentate to monodentate. DFT calculations indicate that the bound ligand is most likely a water or a hydroxide molecule representing a product complex. On the other hand the different soaking treatments did not modify the conformation of other pore regions.</p> </div

Terahertz radiation induces non-thermal structural changes associated with Fröhlich condensation in a protein crystal

Whether long-range quantum coherent states could exist in biological systems, and beyond low-temperature regimes where quantum physics is known to be applicable, has been the subject to debate for decades. It was proposed by Fröhlich that vibrational modes within protein molecules can order and condense into a lowest-frequency vibrational mode in a process similar to Bose-Einstein condensation, and thus that macroscopic coherence could potentially be observed in biological systems. Despite the prediction of these so-called Fröhlich condensates almost five decades ago, experimental evidence thereof has been lacking. Here, we present the first experimental observation of Fröhlich condensation in a protein structure. To that end, and to overcome the challenges associated with probing low-frequency molecular vibrations in proteins(which has hampered understanding of their role in proteins' function), we combined terahertz techniques with a highly sensitive X-ray crystallographic method to visualize low-frequency vibrational modes in the protein structure of hen-egg white lysozyme. We found that 0.4 THz electromagnetic radiation induces non-thermal changes in electron density. In particular, we observed a local increase of electron density in a long α-helix motif consistent with a subtle longitudinal compression of the helix. These observed electron density changes occur at a low absorption rate indicating that thermalization of terahertz photons happens on a micro- to milli-second time scale, which is much slower than the expected nanosecond time scale due to damping of delocalized low frequency vibrations. Our analyses show that the micro- to milli-second lifetime of the vibration can only be explained by Fröhlich condensation, a phenomenon predicted almost half a century ago, yet never experimentally confirmed

Hole2 representations of the 4 fold pore.

<p>(A) Overview of the <i>Bv</i> Bfr 4-fold pore (B) Zoom onto the residues immediately surrounding the 4-fold pore in <i>Bv</i> Bfr and (C) <i>Pa</i> Bfr 4-fold pore with a potassium ion (<i>pink</i>) modeled in the center of the pore (PDB entry 3ISF) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046992#pone.0046992-Weeratunga1" target="_blank">[8]</a>.</p

Interatomic distances in the different <i>Bv</i> Bfr structures compared to the DFT optimized distances from Compounds A and B from Figure S1.

<p>Interatomic distances in the different <i>Bv</i> Bfr structures compared to the DFT optimized distances from Compounds A and B from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046992#pone.0046992.s001" target="_blank">Figure S1</a>.</p

Hole2 representation of the <i>Bv</i> Bfr B-pore.

<p>The side chains of amino acid residues surrounding the constricted region of the pore are also indicated.</p

Multiple sequence alignment.

<p>Multiple sequence alignment of bacterioferritin from Blastochloris viridis (Bv), Rhodobacter sphaeroides (Rs), Rhodobacter capsulatus (Rc), Escherichia coli (Ec), Pseudomonas aeruginosa (Pa), Azotobacter vinelandii (Av), Mycobacterium smegmatis (Ms), Mycobacterium tuberculosis (Mt) and Desulfovibrio desulfuricans (Dd). The more conserved positions are colored darker red. The figure was produced with the program Aline. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046992#pone.0046992-Bond1" target="_blank">[41]</a>.</p