Family of TREMs and TREM-like receptors The most significant accomplishment of last year was the structure solution of the soluble fragment of human TLT-1. We also showed that the soluble variant of this protein exists in vivo, making our finding more relevant. As a side result of our efforts, we developed the first protocol for expression and folding of the functional TLT-1 at the preparative scale. Recently, we have identified a putative ligand of TLT-1 by probing a lysate from human platelets with the His6-tagged-TLT-1. Final confirmation of the ligand's identity is in progress. We are also aiming to crystallize the cytoplasmic domain of TLT-1, which has been already expressed and purified. In another set of experiments, we are attempting to crystallize a complex between the extracellular domain of TLT-1 and the single-chain monoclonal antibody (C10). The antibody has been expressed and purified according to newly developed protocol. Formation and purification of the specific TLT1-C10 complex has been accomplished and crystallization trials are under way. Similar to the one described for TLT-1 strategy has been attempted for a related protein, TREM-2. Currently, we are try to overcome the difficulties related to expression and purification of the extracellular domain of this protein. Structural studies of ASAP1 The main goal of this most recent research exploration is establish the crystallization conditions for the multi-domain fragments of ASAP1 and possibly for a whole enzyme. Following constructs are currently subjected to the crystallization trials: PH-AfrGAP-2 Ank (a.a. 330-725, 330-740), AfrGAP-2 Ank (a.a. 430-725, 440-725), Bar- AfrGAP-2 Ank (a.a. 1-725, 1-740). All proteins are also subjected to a partial proteolysis with thermolysin or the methylation of primary amino groups (Lys, N-terminus) to search for the shorter/modified variants, more amenable for crystallization. Structural studies of isoaspartyl dipeptidase Three X-ray structures of SynA originated from different crystal forms have been determined by members of our Section. In addition to two structures of unliganded SynA, in the third structure we identified the product of enzymatic reaction bound to the active site of the enzyme. The careful analysis of these structures and comparison to related enzymes, revealed several topological features, responsible for discrimination of different substrates. The manuscript, describing our findings was submitted for publication, and this research is considered as completed. Structural and functional studies of GCPII and GCPIII Research focused on studies of GCPII and GCPIII is considered primarily as largely independent activity of Dr. Barinka, who conducts it in addition to his extensive involvement in the main Project of the Section. An important purpose of conducting this work is to establish the project carried by Dr. Barinka as an independent scientist, after completion of his postdoctoral felldowship in the Section. However, the fact that GCPII is an excellent target for prostate cancer imaging and therapy. Prior our work the only structure of unliganded GCPII was determined at the resolution of 3.3 A, which is grossly insufficient for successful development of inhibitors in rational manner. The first major achievement of our Section was a determination of the structures of native, unliganded GCPII and GCPIII at the resolutions significantly exceeding 2 A. Subsequently, we have solved the X-ray structures of 10 complexes between these enzymes and series of novel inhibitors (provided by industrial collaborator, MGI Pharma, Inc., 6611 Tributary Street, Baltimore, MD, USA). Our results form a very solid foundation for development of a new class of potent inhibitors of GCPII/III characterized by very high specificity. The results partially reported in a form of two publications whereas additional three manuscripts are near completion.