Structural studies of gelsolin and binding partners

Gelsolin is composed of six similarly folded domains, G1 through G6. It controls actin dynamics through its calcium-sensitive ability to bind to, sever, and cap F-actin filaments. This project was designed to test and extend current models for gelsolin activity by growing crystals of intact gelsolin or large fragments of gelsolin in protein complexes with its main target, actin, as well as with specialized gelsolin-specific antibody fragments, nanobodies. Gelsolin-specific nanobodies (GsnVHH) may serve to lock in either resting or activated forms of gelsolin structure, but are of intrinsic interest on their own in that they have potential therapeutic value. This thesis describes cloning, expression, purification, activity assays, crystallization details, and solution of the structures of six proteins and protein complexes. 1) Recombinant human G1-G3 bound to natural source rabbit actin (G1-G3:actin). The present structure is to a higher resolution than earlier ones, providing increased confidence in positioning of amino acid side chains and bound Ca²⁺ ions. 2) Isolated, activated gelsolin domain G3. While not previously reported in the literature, this structure is virtually identical to that observed for activated G3 within the G1-G3:actin complex. 3) Activated recombinant human G4-G6. The source material and crystallization conditions are different from those reported elsewhere, but the results confirm that the C-terminal half of gelsolin can be fully activated in the presence of Ca²⁺, even in the absence of actin. 4) and 5) Two crystal structures of GsnVHH11. This nanobody had not previously been crystallized. The two structures differ in the path of the CDR1 loop that is involved in binding gelsolin. 6) GsnVHH9. The results, from different materials and crystallization conditions than previously reported, confirm the validity of the previous structure. Finally, runs using in silico protein-protein docking software suggest possible binding sites for GsnVHH11 and GsnVHH13 on activated G2-G3 and activated G4-G6, respectively.Science, Faculty ofChemistry, Department ofGraduat

Calcium ion exchange in crystalline gelsolin

Gelsolin is a calcium and pH-sensitive modulator of actin filament length. Here, we use X-ray crystallography to examine the extraction and exchange of calcium ions from their binding sites in different crystalline forms of the activated N and C-terminal halves of gelsolin, G1-G3 and G4-G6, respectively. We demonstrate that the combination of calcium and low pH activating conditions do not induce conformational changes in G4-G6 beyond those elicited by calcium alone. EGTA is able to remove calcium ions bound to the type I and type II metal ion-binding sites in G4-G6. Constrained by crystal contacts and stabilized by interdomain interaction surfaces, the gross structure of calcium-depleted G4-G6 remains that of the activated form. However, high-resolution details of changes in the ion-binding sites may represent the initial steps toward restoration of the arrangement of domains found in the calcium-free inactive form of gelsolin in solution. Furthermore, bathing crystals with the trivalent calcium ion mimic, Tb3+, results in anomalous scattering data that permit unequivocal localization of terbium ions in each of the proposed type I and type II ion-binding sites of both halves of gelsolin. In contrast to predictions based on solution studies, we find that no calcium ion is immune to exchange

An ER-directed gelsolin nanobody targets the first step in amyloid formation in a gelsolin amyloidosis mouse model

Hereditary gelsolin amyloidosis is an autosomal dominantly inherited amyloid disorder. A point mutation in the GSN gene (G654A being the most common one) results in disturbed calcium binding by the second gelsolin domain (G2). As a result, the folding of G2 is hampered, rendering the mutant plasma gelsolin susceptible to a proteolytic cascade. Consecutive cleavage by furin and MT1-MMP-like proteases generates 8 and 5 kDa amyloidogenic peptides that cause neurological, ophthalmological and dermatological findings. To this day, no specific treatment is available to counter the pathogenesis. Using GSN nanobody 11 as a molecular chaperone, we aimed to protect mutant plasma gelsolin from furin proteolysis in the trans-Golgi network. We report a transgenic, GSN nanobody 11 secreting mouse that was used for crossbreeding with gelsolin amyloidosis mice. Insertion of the therapeutic nanobody gene into the gelsolin amyloidosis mouse genome resulted in improved muscle contractility. X-ray crystal structure determination of the gelsolin G2:Nb11 complex revealed that Nb11 does not directly block the furin cleavage site. We conclude that nanobodies can be used to shield substrates from aberrant proteolysis and this approach might establish a novel therapeutic strategy in amyloid diseases

Macrophage Matrix Metalloproteinase-12 Dampens Inflammation and Neutrophil Influx in Arthritis

Resolution of inflammation reduces pathological tissue destruction and restores tissue homeostasis. Here, we used a proteomic protease substrate discovery approach, terminal amine isotopic labeling of substrates (TAILS), to analyze the role of the macrophage-specific matrix metalloproteinase-12 (MMP12) in inflammation. In murine peritonitis, MMP12 inactivates antithrombin and activates prothrombin, prolonging the activated partial thromboplastin time. Furthermore, MMP12 inactivates complement C3 to reduce complement activation and inactivates the chemoattractant anaphylatoxins C3a and C5a, whereas iC3b and C3b opsonin cleavage increases phagocytosis. Loss of these anti-inflammatory activities in collagen-induced arthritis in Mmp12−/− mice leads to unresolved synovitis and extensive articular inflammation. Deep articular cartilage loss is associated with massive neutrophil infiltration and abnormal DNA neutrophil extracellular traps (NETs). The NETs are rich in fibrin and extracellular actin, which TAILS identified as MMP12 substrates. Thus, macrophage MMP12 in arthritis has multiple protective roles in countering neutrophil infiltration, clearing NETs, and dampening inflammatory pathways to prepare for the resolution of inflammation

Helix Straightening as an Activation Mechanism in the Gelsolin Superfamily of Actin Regulatory Proteins*

Villin and gelsolin consist of six homologous domains of the gelsolin/cofilin fold (V1–V6 and G1–G6, respectively). Villin differs from gelsolin in possessing at its C terminus an unrelated seventh domain, the villin headpiece. Here, we present the crystal structure of villin domain V6 in an environment in which intact villin would be inactive, in the absence of bound Ca2+ or phosphorylation. The structure of V6 more closely resembles that of the activated form of G6, which contains one bound Ca2+, rather than that of the calcium ion-free form of G6 within intact inactive gelsolin. Strikingly apparent is that the long helix in V6 is straight, as found in the activated form of G6, as opposed to the kinked version in inactive gelsolin. Molecular dynamics calculations suggest that the preferable conformation for this helix in the isolated G6 domain is also straight in the absence of Ca2+ and other gelsolin domains. However, the G6 helix bends in intact calcium ion-free gelsolin to allow interaction with G2 and G4. We suggest that a similar situation exists in villin. Within the intact protein, a bent V6 helix, when triggered by Ca2+, straightens and helps push apart adjacent domains to expose actin-binding sites within the protein. The sixth domain in this superfamily of proteins serves as a keystone that locks together a compact ensemble of domains in an inactive state. Perturbing the keystone initiates reorganization of the structure to reveal previously buried actin-binding sites