Experimental Investigations on the Structure of Yeast Mitochondrial Pyruvate Carriers.

Mitochondrial pyruvate carrier (MPC) transports pyruvate from the cytoplasm into the mitochondrial matrix to participate in the tricarboxylic acid (TCA) cycle, which further generates the energy for the physiological activities of cells. Two interacting subunits, MPC1 and MPC2 or MPC3, form a heterodimer to conduct transport function. However, the structural basis of how the MPC complex transports pyruvate is still lacking. Here, we described the detailed expression and purification procedures to obtain large amounts of yeast MPC1 and MPC2 for structural characterization. The purified yeast MPC1 and MPC2 were reconstituted in dodecylphosphocholine (DPC) micelles and examined using nuclear magnetic resonance (NMR) spectroscopy, showing that both subunits contain three α-helical transmembrane regions with substantial differences from what was predicted by AlphaFold2. Furthermore, the new protocol producing the recombinant MPC2 using modified maltose-binding protein (MBP) with cyanogen bromide (CNBr) cleavage introduced general way to obtain small membrane proteins. These findings provide a preliminary understanding for the structure of the MPC complex and useful guidance for further studies.

DNA-Mediated Assembly of Multispecific Antibodies for T Cell Engaging and Tumor Killing.

Targeting T-cells against cancer cells is a direct means of treating cancer, and has already shown great responses in clinical treatment of B-cell malignancies. A simple way to redirect T-cells to cancer cells is by using multispecific antibody (MsAb) that contains different arms for specifically "grabbing" the T-cells and cancer cells; as such, the T-cells are activated upon target engagement and the killing begins. Here, a nucleic acid mediated protein-protein assembly (NAPPA) approach is implemented to construct a MsAb for T-cell engaging and tumor killing. Anti -CD19 and -CD3 single-chain variable fragments (scFvs) are conjugated to different l-DNAs with sequences that form the Holliday junction, thus allowing spontaneous assembly of homogeneous protein-DNA oligomers containing two anti-CD19 and one anti-CD3 scFvs. The new MsAb shows strong efficacy in inducing Raji tumor cell cytotoxicity in the presence of T-cells with EC50 ≈ 0.2 × 10-9 m; it also suppresses tumor growth in a Raji xenograft mouse model. The data indicates that MsAbs assembled from protein-DNA conjugates are effective macromolecules for directing T-cells for tumor killing. The modular nature of the NAPPA platform allows rapid generation of complex MsAbs from simple antibody fragments, while offering a general solution for preparing antibodies with high-order specificity.

Specific Lipid Binding of Membrane Proteins in Detergent Micelles Characterized by NMR and Molecular Dynamics.

Many membrane proteins bind specifically to lipids as an integral component of their structures. The ability of detergents to support lipid binding is thus an important consideration when solubilizing membrane proteins for structural studies. In particular, the zwitterionic phosphocholine (PC)-based detergents, which have been widely used in solution NMR studies of channels and transporters, are controversial because of their strong solubilization power and thus perceived as more denaturing than nonionic detergents such as the maltosides. Here, we investigate the ability of the mitochondrial ADP/ATP carrier (AAC) to specifically bind cardiolipin, a mitochondrial lipid important for the carrier function, in dodecylphosphocholine (DPC) micelles. We found that in DPC, the AAC specifically binds cardiolipin in a manner consistent with the bound cardiolipins found in the crystal structures of the AAC determined in n-decyl ß-d-maltoside. Our results suggest that PC detergent is compatible with specific lipid binding and that PC detergent mixed with the relevant lipid represents a viable solubilization system for NMR studies of membrane proteins.

Molecular Basis of MgATP Selectivity of the Mitochondrial SCaMC Carrier.

The mitochondrial matrix is the supplier of cellular ATP. The short Ca(2+)-binding mitochondrial carrier (SCaMC) is one of the two mitochondrial carriers responsible for transporting ATP across the mitochondrial inner membrane. While the ADP/ATP carrier (AAC) accounts for the bulk ADP/ATP recycling in the matrix, the function of SCaMC is important for mitochondrial activities that depend on adenine nucleotides, such as gluconeogenesis and mitochondrial biogenesis. A key difference between SCaMC and AAC is that SCaMC selectively transports MgATP whereas AAC only transports free nucleotides. Here, we use a combination of nuclear magnetic resonance experiments and functional mutagenesis to investigate the structural basis of the MgATP selectivity in SCaMC. Our data revealed an MgATP binding site inside the transporter cavity, while identifying an aspartic acid residue that plays an important role in the higher selectivity for MgATP over free ATP.

Substrate-modulated ADP/ATP-transporter dynamics revealed by NMR relaxation dispersion.

The ADP/ATP carrier (AAC) transports ADP and ATP across the inner mitochondrial membrane. Unlike most transporters, which have two-fold direct or inverted quasisymmetry, AAC has apparent three-fold rotational symmetry. Further, its transport rate is relatively fast for transporters that carry large solutes. Here, we study the yeast AAC carrier 3 by obtaining comprehensive NMR relaxation dispersion measurements, which provide residue-specific information on the protein's conformational exchange. Our data indicate that AAC is predominantly in the cytosol-facing open state and converts to a sparsely populated state in an asymmetric manner despite its three-fold structural symmetry. Binding of the substrate ADP substantially increases the rate of conformational exchange, whereas the inhibitor CATR slows the exchange. These results suggest that although the transporter catalyzes the translocation of substrate the substrate also facilitates interconversion between alternating states, and this interconversion may be relevant to the transport function.