Youg Raj Thaker

Specific motifs of the V-ATPase a2-subunit isoform interact with catalytic and regulatory domains of ARNO.

We have previously shown that the V-ATPase a2-subunit isoform interacts specifically, and in an intra-endosomal acidification-dependent manner, with the Arf-GEF ARNO. In the present study, we examined the molecular mechanism of this interaction using synthetic peptides and purified recombinant proteins in protein-association assays. In these experiments, we revealed the involvement of multiple sites on the N-terminus of the V-ATPase a2-subunit (a2N) in the association with ARNO. While six a2N-derived peptides interact with wild-type ARNO, only two of them (named a2N-01 and a2N-03) bind to its catalytic Sec7-domain. However, of these, only the a2N-01 peptide (MGSLFRSESMCLAQLFL) showed specificity towards the Sec7-domain compared to other domains of the ARNO protein. Surface plasmon resonance kinetic analysis revealed a very strong binding affinity between this a2N-01 peptide and the Sec7-domain of ARNO, with dissociation constant KD=3.44x10(-7) M, similar to the KD=3.13x10(-7) M binding affinity between wild-type a2N and the full-length ARNO protein. In further pull-down experiments, we also revealed the involvement of multiple sites on ARNO itself in the association with a2N. However, while its catalytic Sec7-domain has the strongest interaction, the PH-, and PB-domains show much weaker binding to a2N. Interestingly, an interaction of the a2N to a peptide corresponding to ARNOs PB-domain was abolished by phosphorylation of ARNO residue Ser392. The 3D-structures of the non-phosphorylated and phosphorylated peptides were resolved by NMR spectroscopy, and we have identified rearrangements resulting from Ser392 phosphorylation. Homology modeling suggests that these alterations may modulate the access of the a2N to its interaction pocket on ARNO that is formed by the Sec7 and PB-domains. Overall, our data indicate that the interaction between the a2-subunit of V-ATPase and ARNO is a complex process involving various binding sites on both proteins. Importantly, the binding affinity between the a2-subunit and ARNO is in the same range as those previously reported for the intramolecular association of subunits within V-ATPase complex itself, indicating an important cell biological role for the interaction between the V-ATPase and small GTPase regulatory proteins.

TCR and CD28 activate the transcription factor NF-κB in T-cells via distinct adaptor signaling complexes.

Highlights • CD28 and TCR receptors use independent pathways to regulate NF-κB activation in T-cells.• CD28 mediated NF-κB activation is dependent on the YMN-FM site for GRB-2 adaptor binding.• The adaptors ADAP and SKAP1 are dispensable for direct CD28 activation of NF-κB.• TCR driven NF-κB activation requires adaptor ADAP expression.

Assembly of subunit d (Vma6p) and G (Vma10p) and the NMR solution structure of subunit G (G(1-59)) of the Saccharomyces cerevisiae V(1)V(O) ATPase.

Understanding the structural traits of subunit G is essential, as it is needed for V(1)V(O) assembly and function. Here solution NMR of the recombinant N- (G(1-59)) and C-terminal segment (G(61-114)) of subunit G, has been performed in the absence and presence of subunit d of the yeast V-ATPase. The data show that G does bind to subunit d via its N-terminal part, G(1-59) only. The residues of G(1-59) involved in d binding are Gly7 to Lys34. The structure of G(1-59) has been solved, revealing an alpha-helix between residues 10 and 56, whereby the first nine- and the last three residues of G(1-59) are flexible. The surface charge distribution of G(1-59) reveals an amphiphilic character at the N-terminus due to positive and negative charge distribution at one side and a hydrophobic surface on the opposite side of the structure. The C-terminus exhibits a strip of negative residues. The data imply that G(1-59)-d assembly is accomplished by hydrophobic interactions and salt-bridges of the polar residues. Based on the recently determined NMR structure of segment E(18-38) of subunit E of yeast V-ATPase and the presently solved structure of G(1-59), both proteins have been docked and binding epitopes have been analyzed.

Spectroscopical identification of residues of subunit G of the yeast V-ATPase in its connection with subunit E

A critical point in the V1 sector and entire V1VO complex is the interaction of stalk subunits G (Vma10p) and E (Vma4p). Previous work, using precipitation assays, has shown that both subunits form a complex. In this work, we have analysed the N-terminal segment of subunit G (G1–59) of the V1VO ATPase from Saccharomyces cerevisiae by using nuclear magnetic resonance (NMR) spectroscopy. Analyses of 1H-15N heteronuclear single quantum coherence (HSQC) spectra of G1–59 in the absence and presence of the N-terminal peptides E1–18 and E18–38 as well as the produced and purified C-terminal segment (E39–233) shows specific interactions only with the peptide fragment E18–38. The binding of this peptide occurs via the residues M1, V2, S3, and K5 as well for V22, S23, K24, A25 and R26 of G1–59. The specific E18–38/G1–59 binding has been confirmed by fluorescence correlation spectroscopy data. The E18–38 peptide has been studied by CD spectroscopy and NMR. The 3D structure of this peptide adopts a stable helix-hinge-helix formation in solution. A model structure of the E18–38/G1–59 complex reveals the orientation of E18–38 relative to G1–59 via salt-bridges of the polar residues and van der Waals forces at the very N-terminus of both segments.

NMR solution structure of subunit E (fragment E1–69) of the Saccharomyces cerevisiae V1VO ATPase

The N-terminus of V-ATPase subunit E has been shown to associate with the subunits C, G and H, respectively. To understand the assembly of E with its neighboring subunits as well as its N-terminal structure, the N-terminal region, E1-69, of the Saccharomyces cerevisiae V-ATPase subunit E was expressed and purified. The solution structure of E1-69 was determined by NMR spectroscopy. The protein is 90.3 Å in length and forms an á-helix between the residues 12–68. The molecule is amphipathic with hydrophobic residues at the N-terminus, predicted to interact with subunit C. The polar epitopes of E1–69 are discussed as areas interacting with subunits G and H.

Solution structure of subunit F (Vma7p) of the eukaryotic V1VO ATPase from Saccharomyces cerevisiae derived from SAXS and NMR spectroscopy

Vacuolar ATPases use the energy derived from ATP hydrolysis, catalyzed in the A(3)B(3) sector of the V(1) ATPase to pump protons via the membrane-embedded V(O) sector. The energy coupling between the two sectors occurs via the so-called central stalk, to which subunit F does belong. Here we present the first low resolution structure of recombinant subunit F (Vma7p) of a eukaryotic V-ATPase from Saccharomyces cerevisiae, analyzed by small angle X-ray scattering (SAXS). The protein is divided into a 5.5nm long egg-like shaped region, connected via a 1.5nm linker to a hook-like segment at one end. Circular dichroism spectroscopy revealed that subunit F comprises of 43% α-helix, 32% β-sheet and a 25% random coil arrangement. To determine the localization of the N- and C-termini in the protein, the C-terminal truncated form of F, F(1-94) was produced and analyzed by SAXS. Comparison of the F(1-94) shape with the one of subunit F showed the missing hook-like region in F(1-94), supported by the decreased D(max) value of F(1-94) (7.0nm), and indicating that the hook-like region consists of the C-terminal residues. The NMR solution structure of the C-terminal peptide, F(90-116), was solved, displaying an α-helical region between residues 103 and 113. The F(90-116) solution structure fitted well in the hook-like region of subunit F. Finally, the arrangement of subunit F within the V(1) ATPase is discussed.

Association of the eukaryotic V1VO ATPase subunits a with d and d with A

MINT‐7012028: d (uniprotkb:P32366) binds (MI:0407) to a (uniprotkb:P32563) by surface plasmon resonance (MI:0107)