Kindlin-3 mediates integrin αLß2 outside-in signaling, and it interacts with scaffold protein receptor for activated-C kinase 1 (RACK1).

Integrins are heterodimeric type I membrane cell adhesion molecules that are involved in many biological processes. Integrins are bidirectional signal transducers because their cytoplasmic tails are docking sites for cytoskeletal and signaling molecules. Kindlins are cytoplasmic molecules that mediate inside-out signaling and activation of the integrins. The three kindlin paralogs in humans are kindlin-1, -2, and -3. Each of these contains a 4.1-ezrin-radixin-moesin (FERM) domain and a pleckstrin homology domain. Kindlin-3 is expressed in platelets, hematopoietic cells, and endothelial cells. Here we show that kindlin-3 is involved in integrin αLß2 outside-in signaling. It also promotes micro-clustering of integrin αLß2. We provide evidence that kindlin-3 interacts with the receptor for activated-C kinase 1 (RACK1), a scaffold protein that folds into a seven-blade propeller. This interaction involves the pleckstrin homology domain of kindlin-3 and blades 5-7 of RACK1. Using the SKW3 human T lymphoma cells, we show that integrin αLß2 engagement by its ligand ICAM-1 promotes the association of kindlin-3 with RACK1. We also show that kindlin-3 co-localizes with RACK1 in polarized SKW3 cells and human T lymphoblasts. Our findings suggest that kindlin-3 plays an important role in integrin αLß2 outside-in signaling.

Binding of CDR-derived peptides is mechanistically different from that of high-affinity parental antibodies.

We present data that reveal crucial differences between the binding mode of anti-gastrin17 (G17, pyroEGPWLEEEEEAYGWMDF-NH(2)) monoclonal antibodies (mAbs) and their CDR-derived synthetic binders (SBs) with G17. The mAbs recognize the N-terminal sequence of G17 (pyroEGPWL) with nanomolar affinity and high sequence selectivity. Molecular simulations suggest that G17 recognition is based primarily on a multitude of weak antibody-ligand interactions (H-bonding, van der Waals, etc.) inside a structurally well-defined cleft-like binding pocket. Relatively small structural changes (e.g. G-2 to A for G17) have a drastic impact on affinity, which is characteristic for antibody-like binding. In contrast, SBs recognize various sequences, including G17-unrelated targets with affinities of 1:1 complexes estimated in the 0.1-1.0 mM range. In most cases however, the G17/SB complex stoichiometries are not well-defined, giving rise to multimer aggregate formation with high apparent complex stabilities. Mutational studies on both G17 and SBs reveal the importance of positively charged (K/R) and aromatic residues (W/Y/F) for G17/SB complex formation. We propose that the synthetic binders use combinations of electrostatic, hydrophobic, and/or cation-π interactions in a variety of ways due to their intrinsic flexibility. This may also be the reason for their relatively low target specificity. We speculate that our findings are of general relevance, in showing that high-affinity mAbs do not necessarily provide the optimal basis for functional mimics design.

Modulation of the Vault Protein-Protein Interaction for Tuning of Molecular Release.

Vaults are naturally occurring ovoid nanoparticles constructed from a protein shell that is composed of multiple copies of major vault protein (MVP). The vault-interacting domain of vault poly(ADP-ribose)-polymerase (INT) has been used as a shuttle to pack biomolecular cargo in the vault lumen. However, the interaction between INT and MVP is poorly understood. It is hypothesized that the release rate of biomolecular cargo from the vault lumen is related to the interaction between MVP and INT. To tune the release of molecular cargos from the vault nanoparticles, we determined the interactions between the isolated INT-interacting MVP domains (iMVP) and wild-type INT and compared them to two structurally modified INT: 15-amino acid deletion at the C terminus (INTΔC15) and histidine substituted at the interaction surface (INT/DSA/3 H) to impart a pH-sensitive response. The apparent affinity constants determined using surface plasmon resonance (SPR) biosensor technology are 262 ± 4 nM for iMVP/INT, 1800 ± 160 nM for iMVP/INTΔC15 at pH 7.4. The INT/DSA/3 H exhibits stronger affinity to iMVP (K Dapp = 24 nM) and dissociates at a slower rate than wild-type INT at pH 6.0.

Binding of TCR multimers and a TCR-like antibody with distinct fine-specificities is dependent on the surface density of HLA complexes.

Class I Major Histocompatibility Complex (MHC) molecules evolved to sample degraded protein fragments from the interior of the cell, and to display them at the surface for immune surveillance by CD8(+) T cells. The ability of these lymphocytes to identify immunogenic peptide-MHC (pMHC) products on, for example, infected hepatocytes, and to subsequently eliminate those cells, is crucial for the control of hepatitis B virus (HBV). Various protein scaffolds have been designed to recapitulate the specific recognition of presented antigens with the aim to be exploited both diagnostically (e.g. to visualize cells exposed to infectious agents or cellular transformation) and therapeutically (e.g. for the delivery of drugs to compromised cells). In line with this, we report the construction of a soluble tetrameric form of an αß T cell receptor (TCR) specific for the HBV epitope Env(183-191) restricted by HLA-A*02:01, and compare its avidity and fine-specificity with a TCR-like monoclonal antibody generated against the same HLA target. A flow cytometry-based assay with streptavidin-coated beads loaded with Env(183-191)/HLA-A*02:01 complexes at high surface density, enabled us to probe the specific interaction of these molecules with their cognate pMHC. We demonstrate that the TCR tetramer has similar avidity for the pMHC as the antibody, but they differ in their fine-specificity, with only the TCR tetramer being capable of binding both natural variants of the Env(183-191) epitope found in HBV genotypes A/C/D (187Arg) and genotype B (187Lys). Collectively, the results highlight the promiscuity of our soluble TCR, which could be an advantageous feature when targeting cells infected with a mutation-prone virus, but that binding of the soluble oligomeric TCR relies considerably on the surface density of the presented antigen.