SUMMARY UCS proteins, such as UNC-45, influence muscle contraction and other myosin-dependent motile processes. We report the first x-ray crystal structure of a UCS domain-containing protein, the UNC-45 myosin chaperone from Drosophila melanogaster (DmUNC-45). The structure reveals that the Central and UCS domains form a contiguous arrangement of seventeen consecutive helical layers that arrange themselves into five discrete armadillo repeat subdomains. Small-angle x-ray scattering data suggest that free DmUNC-45 adopts an elongated conformation and exhibits flexibility in solution. Protease sensitivity maps to a conserved loop that contacts the most carboxy-terminal UNC-45 armadillo repeat sub-domain. Amino acid conservation across diverse UCS proteins maps to one face of this carboxy-terminal sub-domain and the majority of mutations that affect myosin-dependent cellular activities lie within or around this region. Our crystallographic, biophysical, and biochemical analyses suggest that DmUNC-45 function is afforded by its flexibility and by structural integrity of its UCS domain.

BackgroundLysophosphatidic acid (LPA) is a bioactive phospholipid with a potentially causative role in neurotrauma. Blocking LPA signaling with the LPA-directed monoclonal antibody B3/Lpathomab is neuroprotective in the mouse spinal cord following injury.FindingsHere we investigated the use of this agent in treatment of secondary brain damage consequent to traumatic brain injury (TBI). LPA was elevated in cerebrospinal fluid (CSF) of patients with TBI compared to controls. LPA levels were also elevated in a mouse controlled cortical impact (CCI) model of TBI and B3 significantly reduced lesion volume by both histological and MRI assessments. Diminished tissue damage coincided with lower brain IL-6 levels and improvement in functional outcomes.ConclusionsThis study presents a novel therapeutic approach for the treatment of TBI by blocking extracellular LPA signaling to minimize secondary brain damage and neurological dysfunction.

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Lysophosphatidic acid (LPA) is a common product of glycerophospholipid metabolism and an important mediator of signal transduction. Aberrantly high LPA concentrations accompany multiple disease states. One potential approach for treatment of these diseases, therefore, is the therapeutic application of antibodies that recognize and bind LPA as their antigen. We have determined the x-ray crystal structure of an anti-LPA antibody (LT3015) Fab fragment in its antigen-free form to 2.15 Å resolution and in complex with two LPA isotypes (14:0 and 18:2) to resolutions of 1.98 and 2.51 Å, respectively. The variable complementarity determining region (CDR) loops at the antigen binding site adopt nearly identical conformations in the free and antigen-bound crystal structures. The crystallographic models reveal that the LT3015 antibody employs both heavy and light chain CDR loops to create a network of eight hydrogen bonds with the glycerophosphate head group of its LPA antigen. The head group is almost completely excluded from contact with solvent, while the hydrocarbon tail is partially solvent exposed. In general, mutation of amino acid residues at the antigen binding site disrupts LPA binding. However, the introduction of particular mutations chosen strategically based upon the structures can positively influence LPA binding affinity. Finally, these structures elucidate the exquisite specificity demonstrated by an anti-lipid antibody for binding a structurally simple and seemingly unconstrained target molecule.