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Development of novel protein-based drugs such as antibodies or immunocytokines and initial validation of target binding typically occurs in 2D settings with e.g., surface plasmon resonance or reflection interferometry using antigen-coated planar sensors. However, a versatile tool to assess binding characteristics of tumour-targeting therapeutics where specific biochemical interactions can be monitored in real-time and in a more realistic 3D interaction setting is currently lacking. Here, we report on the development of versatile small optical 3D biosensors using protein G-coated spherical microparticles based on Whispering Gallery Modes (WGMs). These sensors allowed for an oriented immobilization of theoretically any immunoglobulin G (IgG) and IgG crystallizable fragment domain (Fc)-tagged protein and have been carefully optimized for specific detection of antigen-antibody interactions, as illustrated using the Epithelial Growth Factor Receptor (EGFR) antibody Cetuximab and Program Death Ligand 1 (PD-L1) antibody Atezolizumab. When both ligand and analyte contained an Fc-fragment, protein G binding capacity saturation followed by a “blocking” step with an irrelevant IgG enabled the label-free detection of Fc-tagged antigens with corresponding cognate ligands, as identified using EGFR-Fc, PD-L1-Fc and different members from the tumour necrosis factor receptor family, such as CD27-Fc and 4−1BB-Fc. Thus, WGMs and developed protein G-coated biosensors provide a widely applicable tool to evaluate binding of immunotherapeutics to their targets on the sensor surface, imitating cell surface properties.

Protein phosphatase 5 is involved in the regulation of kinases and transcription factors. The dephosphorylation activity is modulated by the molecular chaperone Hsp90, which binds to the TPR-domain of protein phosphatase 5. This interaction is dependent on the C-terminal MEEVD motif of Hsp90. We show that C-terminal Hsp90 fragments differ in their regulation of the phosphatase activity hinting to a more complex interaction. Also hydrodynamic parameters from analytical ultracentrifugation and small-angle X-ray scattering data suggest a compact structure for the Hsp90-protein phosphatase 5 complexes. Using crosslinking experiments coupled with mass spectrometric analysis and structural modelling we identify sites, which link the middle/C-terminal domain interface of C. elegans Hsp90 to the phosphatase domain of the corresponding kinase. Studying the relevance of the domains of Hsp90 for turnover of native substrates we find that ternary complexes with the glucocorticoid receptor (GR) are cooperatively formed by full-length Hsp90 and PPH-5. Our data suggest that the direct stimulation of the phosphatase activity by C-terminal Hsp90 fragments leads to increased dephosphorylation rates. These are further modulated by the binding of clients to the N-terminal and middle domain of Hsp90 and their presentation to the phosphatase within the phosphatase-Hsp90 complex.

In the phototransduction pathway of rhodopsin, the metarhodopsin (Meta) III retinal storage form arises from the active G-protein binding Meta II by a slow spontaneous reaction through the Meta I precursor or by light absorption and photoisomerization, respectively. Meta III is a side product of the Meta II decay path and holds its retinal in the original binding site, with the Schiff base bond to the apoprotein reprotonated as in the dark ground state. It thus keeps the retinal away from the regeneration pathway in which the photolyzed all-trans-retinal is released. This study was motivated by our recent observation that Meta III remains stable for hours in membranes devoid of regulatory proteins, whereas it decays much more rapidly in situ. We have now explored the possibility of regulated formation and decay of Meta III, using intrinsic opsin tryptophan fluorescence and UV-visible and Fourier transform infrared spectroscopy. We find that a rapid return of Meta III into the regeneration pathway is triggered by the G-protein transducin (Gt). Depletion of the retinal storage is initiated by a novel direct bimolecular interaction of Gt with Meta III, which was previously considered inactive. Gt thereby induces the transition of Meta III into Meta II, so that the retinylidene bond to the apoprotein can be hydrolyzed, and the retinal can participate again in the normal retinoid cycle. Beyond the potential significance for retinoid metabolism, this may provide the first example of a G-protein-catalyzed conversion of a receptor.

Nef is the viral gene product employed by the majority of primate lentiviruses to overcome restriction by tetherin (BST-2 or CD317), an interferon-inducible transmembrane protein that inhibits the detachment of enveloped viruses from infected cells. Although the mechanisms of tetherin antagonism by HIV-1 Vpu and HIV-2 Env have been investigated in detail, comparatively little is known about tetherin antagonism by SIV Nef. Here we demonstrate a direct physical interaction between SIV Nef and rhesus macaque tetherin, define the residues in Nef required for tetherin antagonism, and show that the anti-tetherin activity of Nef is dependent on clathrin-mediated endocytosis. SIV Nef co-immunoprecipitated with rhesus macaque tetherin and the Nef core domain bound directly to a peptide corresponding to the cytoplasmic domain of rhesus tetherin by surface plasmon resonance. An analysis of alanine-scanning substitutions identified residues throughout the N-terminal, globular core and flexible loop regions of Nef that were required for tetherin antagonism. Although there was significant overlap with sequences required for CD4 downregulation, tetherin antagonism was genetically separable from this activity, as well as from other Nef functions, including MHC class I-downregulation and infectivity enhancement. Consistent with a role for clathrin and dynamin 2 in the endocytosis of tetherin, dominant-negative mutants of AP180 and dynamin 2 impaired the ability of Nef to downmodulate tetherin and to counteract restriction. Taken together, these results reveal that the mechanism of tetherin antagonism by Nef depends on a physical interaction between Nef and tetherin, requires sequences throughout Nef, but is genetically separable from other Nef functions, and leads to the removal of tetherin from sites of virus release at the plasma membrane by clathrin-mediated endocytosis., Author Summary Tetherin (BST-2, CD317 or HM1.24) is an interferon-inducible cellular restriction factor that prevents the release of enveloped viruses from infected cells. Human and simian immunodeficiency viruses have evolved to use different viral proteins to overcome the anti-viral effects of tetherin. Whereas HIV-1 Vpu and HIV-2 Env counteract human tetherin, most SIVs use the accessory protein Nef to counteract tetherin in their non-human primate hosts. Here we show that the mechanism of tetherin antagonism by SIV Nef involves a direct physical interaction between the core domain of Nef and the cytoplasmic domain of tetherin, which results in the removal of tetherin from sites of virus assembly and release on the cell surface by a mechanism that depends on clathrin and dynamin 2. The Nef-mediated internalization of tetherin leads to the accumulation of tetherin within lysosomal compartments, suggesting that, similar to CD4− and MHC I-downregulation, Nef promotes the lysosomal degradation of tetherin.

Rhodopsin bears 11-cis-retinal covalently bound by a protonated Schiff base linkage. 11-cis/all-trans isomerization, induced by absorption of green light, leads to active metarhodopsin II, in which the Schiff base is intact but deprotonated. The subsequent metabolic retinoid cycle starts with Schiff base hydrolysis and release of photolyzed all-trans-retinal from the active site and ends with the uptake of fresh 11-cis-retinal. To probe chromophore-protein interaction in the active state, we have studied the effects of blue light absorption on metarhodopsin II using infrared and time-resolved UV-visible spectroscopy. A light-induced shortcut of the retinoid cycle, as it occurs in other retinal proteins, is not observed. The predominantly formed illumination product contains all-trans-retinal, although the spectra reflect Schiff base reprotonation and protein deactivation. By its kinetics of formation and decay, its low temperature photointermediates, and its interaction with transducin, this illumination product is identified as metarhodopsin III. This species is known to bind all-trans-retinal via a reprotonated Schiff base and forms normally in parallel to retinal release. We find that its generation by light absorption is only achieved when starting from active metarhodopsin II and is not found with any of its precursors, including metarhodopsin I. Based on the finding of others that metarhodopsin III binds retinal in all-trans-C(15)-syn configuration, we can now conclude that light-induced formation of metarhodopsin III operates by Schiff base isomerization ("second switch"). Our reaction model assumes steric hindrance of the retinal polyene chain in the active conformation, thus preventing central double bond isomerization.

The T cell antigen receptor (TCR) is a cell surface protein complex that binds peptide antigen fragments presented by the Major Histocompatibility Complex (MHC). The interaction of TCR with MHC leads to T cell activation and initiation of an immune response,1 but the molecular details of the transmembrane signaling process remain unknown. TCRζ is the 115 amino acid long cytosolic signaling domain of the CD3-T cell receptor complex that plays a central role in this process. TCRζ is predominantly unstructured2 and carries several copies of the immunoreceptor tyrosine-based activation motif (ITAM). ITAMs become phosphorylated upon receptor engagement, constituting an early and obligatory event in the signaling cascade that blocks lipid association and lipid-associated conformational rearrangements3 of TCRζ. We present a characterization of the affinity of TCRζ to tethered lipid bilayers by SPR and a structural study of its association with such membranes using neutron reflectometry (NR). We studied the binding of unphosphorylated TCRζ to highly charged stBLMs rich in anionic phosphatidylserine (PS) or phosphatidylglycerol (PG) and observed affinity constants of Kd ≈ 10 μM. Minute amounts of phosphoinositides (PI(4,5)P2) increase the membrane affinity of the protein by more than 10-fold. An NR characterization of TCRζ in the membrane-associated state shows the major portion of the protein interfacially associated with the bilayer surface and a minor protein penetrating the bilayer deeply. This is consistent with a model in which an α-helical structure is aligned on the membrane surface that may be connected via a hinge with another α-helical segment inserted into the bilayer.[1] Call, M.E., and Wucherpfennig, K.W., Annu. Rev. Immunol. 23:101 (2005).[2] Sigalov, A.B., et al., Biochemistry 45:15731 (2006).[3] Aivazian, D.A., et al., Nat. Struct. Biol. 7:1023 (2000).