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The twin arginine translocase (Tat) exports folded proteins across bacterial membranes. The putative pore-forming or membrane-weakening component (TatA

Pinholin S2168 triggers the lytic cycle of bacteriophage φ21 in infected Escherichia coli. Activated transmembrane dimers oligomerize into small holes and uncouple the proton gradient. Transmembrane domain 1 (TMD1) regulates this activity, while TMD2 is postulated to form the actual “pinholes.” Focusing on the TMD2 fragment, we used synchrotron radiation-based circular dichroism to confirm its α-helical conformation and transmembrane alignment. Solid-state 15N-NMR in oriented DMPC bilayers yielded a helix tilt angle of τ = 14°, a high order parameter (Smol = 0.9), and revealed the azimuthal angle. The resulting rotational orientation places an extended glycine zipper motif (G40xxxS44xxxG48) together with a patch of H-bonding residues (T51, T54, N55) sideways along TMD2, available for helix–helix interactions. Using fluorescence vesicle leakage assays, we demonstrate that TMD2 forms stable holes with an estimated diameter of 2 nm, as long as the glycine zipper motif remains intact. Based on our experimental data, we suggest structural models for the oligomeric pinhole (right-handed heptameric TMD2 bundle), for the active dimer (right-handed Gly-zipped TMD2/TMD2 dimer), and for the full-length pinholin protein before being triggered (Gly-zipped TMD2/TMD1-TMD1/TMD2 dimer in a line).

Pinholin S(21)68 triggers the lytic cycle of bacteriophage φ21 in infected Escherichia coli. Activated transmembrane dimers oligomerize into small holes and uncouple the proton gradient. Transmembrane domain 1 (TMD1) regulates this activity, while TMD2 is postulated to form the actual “pinholes.” Focusing on the TMD2 fragment, we used synchrotron radiation-based circular dichroism to confirm its α-helical conformation and transmembrane alignment. Solid-state (15)N-NMR in oriented DMPC bilayers yielded a helix tilt angle of τ = 14°, a high order parameter (S(mol) = 0.9), and revealed the azimuthal angle. The resulting rotational orientation places an extended glycine zipper motif (G(40)xxxS(44)xxxG(48)) together with a patch of H-bonding residues (T(51), T(54), N(55)) sideways along TMD2, available for helix–helix interactions. Using fluorescence vesicle leakage assays, we demonstrate that TMD2 forms stable holes with an estimated diameter of 2 nm, as long as the glycine zipper motif remains intact. Based on our experimental data, we suggest structural models for the oligomeric pinhole (right-handed heptameric TMD2 bundle), for the active dimer (right-handed Gly-zipped TMD2/TMD2 dimer), and for the full-length pinholin protein before being triggered (Gly-zipped TMD2/TMD1-TMD1/TMD2 dimer in a line).

Rapid growth in the therapeutic antibody market leads to a drastic development of antibody engineering to optimize biophysical properties. One of the main focuses of biopharmaceutical researches was the expansion of different approaches for affinity maturation of antibodies to enhance biological activity. Adalimumab (D2E7) is an anti-tumor necrosis factor alpha (TNFα) antibody used in the treatment of some autoimmune disorders like rheumatoid arthritis, psoriasis, etc. In this study, by engineering complementary determining regions (CDRs) of D2E7 antibody, we produced a new variant of the antibody with improved affinity and potency to TNFα. We designed four D2E7 mutants that harbored a single point mutation in their CDRs. The native antibody, lc-A94K, lc-A50Y, lc-A33R, and lc-A92R mutant models were transiently produced and characterized. Data showed mutation of ALA to LYS in CDR3 of D2E7 antibody generated new hydrogen bonds with TNFα, thus the lc-A94K model revealed significantly higher binding activity (EC50) and kinetic affinity (KD) to its antigen, in comparison to the wild antibody. Moreover, this model was found to have significantly stronger biological activity (IC50) to activate antibody-dependent cell-mediated cytotoxicity in the mouse fibroblast L929 cells. Secondary structure analysis demonstrated the mutation has no inappropriate conformational impact on the beta and alpha structures of lc-A94K antibody. In conclusion, our study revealed that it is possible to manipulate antibody’s CDRs to increase affinity and potency by single point mutation and also preserve basic structure of the original antibody during CDRs engineering to avoid adverse effects of CDRs mutations on specificity and stability.

We propose a concept for the folding and self- assembly of the pore-forming TatA complex from the Twin-arginine translocase and of other mem- brane proteins based on electrostatic ''charge zippers.'' Each subunit of TatA consists of a trans- membrane segment, an amphiphilic helix (APH), and a C-terminal densely charged region (DCR). The sequence of charges in the DCR is complemen- tary to the charge pattern on the APH, suggesting that the protein can be ''zipped up'' by a ladder of seven salt bridges. The length of the resulting hairpin matches the lipid bilayer thickness, hence a trans- membrane pore could self-assemble via intra- and intermolecular salt bridges. The steric feasibility was rationalized by molecular dynamics simulations, and experimental evidence was obtained by moni- toring the monomer-oligomer equilibrium of specific charge mutants. Similar ''charge zippers'' are pro- posed for other membrane-associated proteins, e.g., the biofilm-inducing peptide TisB, the human antimicrobial peptide dermcidin, and the pestiviral ERNS protein.

The twin arginine translocation (Tat) system can transport fully folded proteins, including their cofactors, across bacterial and thylakoid membranes. The Tat system of Bacillus subtilis that serves to export the phosphodiesterase (PhoD) consists of only two membrane proteins, TatA(d) and TatC(d). The larger component TatC(d) has a molecular weight of 28 kDa and several membrane-spanning segments. This protein has been expressed in Escherichia coli and purified in sufficient amounts for structure analysis by circular dichroism (CD) and NMR spectroscopy. TatC(d) was reconstituted in detergent micelles and in lipid bilayers for CD analysis in solution and in macroscopically oriented samples, to examine the stability of the protein. Suitable protocols and model membrane systems have been established, by which TatC(d) maintains the level of helicity close to theoretically predicted, and its transmembrane alignment could been verified.

The twin-arginine-translocase (Tat) can transport proteins in their folded state across bacterial or thylakoid membranes. In Bacillus subtilis the Tat-machinery consists of only two integral (inner) membrane proteins, TatA and TatC. Multiple copies of TatA are supposed to form the transmembrane channel, but little structural data is available on this 70-residue component. We used a multi-construct approach for expressing several characteristic fragments of TatA(d), to determine their individual structures and to cross-validate them comprehensively within the architecture of the full-length protein. Here, we report the design, high-yield expression, detergent-aided purification and lipid-reconstitution of five constructs of TatA(d), overcoming difficulties associated with the very different hydrophobicities and sizes of these membrane protein fragments. Circular dichroism (CD) and oriented CD (OCD) were used to determine their respective conformations and alignments in suitable, negatively charged phospholipid bilayers. CD spectroscopy showed an N-terminal alpha-helix, a central helical stretch, and an unstructured C-terminus, thus proving the existence of these secondary structures in TatA(d) for the first time. The OCD spectra demonstrated a transmembrane orientation of the N-terminal alpha-helix and a surface alignment of the central amphiphilic helix in lipid bilayers, thus supporting the postulated topology model and function of TatA as a transmembrane channel.

Transmembrane helix–helix interactions mediate the folding and assembly of membrane proteins. Recognition motifs range from GxxxG and leucine zippers to polar side chains and salt bridges. Some canonical membrane proteins contain local charge clusters that are important for folding and function, and which have to be compatible with a stable insertion into the bilayer via the translocon. Recently, the electrostatic “charge zipper” has been described as another kind of assembly motif. The protein sequences exhibit a quasi-symmetrical pattern of complementary charges that can form extended ladders of salt bridges. Such segments can insert reversibly into membranes, or even translocate across them. Nature uses charge zippers in transport processes, and they can also be adapted in the design of cell-penetrating carriers.

Many vital cellular processes, such as protein translocation, proton transport or molecular recognition, are mediated by self assembling membrane proteins. We have investigated the Twin-arginine translocase (TatA) complex, which forms transient pores through which proteins are translocated through the membrane. We postulated that complex formation is electrostatically driven by formation of salt bridges between amphiphilic transmembrane segments of the individual monomers and developed a structure-based model for this process[1].We studied the formation of oligomers of different sizes by structure-based[2] MD simulations in combination with NMR constraints and a hydrophobic-slab implicit membrane model. Starting from isolated monomers, distributed far apart from each other, we observed the formation of stable TatA oligomers on the basis of the postulated interactions. The dimensions of the resulting TatA complex agreed well with experimental electron microscopy measurements[3] and the postulated interactions were confirmed by subsequent mutation studies.[1] T. Walther, C. Gottselig, S. Grage, M. Wolf, A. Vargiu, Marco J. Klein, S. Vollmer, S. Prock, M. Hartmann, S. Afonin, E. Stockwald, H. Heinzmann, W Wenzel, P. Ruggerone, A. Ulrich. Folding and self-assembly of the TatA translocation pore based on a novel charge zipper mechanism, Cell (accepted)[2] Whitford, P. C. et al. An all-atom structure-based potential for proteins: Bridging minimal models with all-atom empirical forcefields. Proteins 75, 430-441 (2009).[3] Gohlke, U. et al. The TatA component of the twin-arginine protein transport system forms channel complexes of variable diameter. Proc. Natl. Acad. Sci. U.S.A. 102, 10482-10486 (2005).

Cells have developed sophisticated transport machineries to allow proteins to cross membrane barriers. In bacteria, the twin arginine translocase (Tat) can translocate proteins even in the folded state. In B. subtilis, a membrane protein complex consisting of two subunits, TatA and TatC, is responsible for the Tat translocation process. TatA is believed to form a nanometer size pore trough which the protein is transported, whereas TatC is involved in recognition of the target protein signal peptide.View Large Image | View Hi-Res Image | Download PowerPoint SlideTo get insight into the mechanism of the Tat translocation in B. subtilis, we studied the structure of the pore-forming subunit TatA and the pore assembly pursuing two complementary experimental approaches. The structure of individual TatA monomers in membranes or membrane-mimetic environments was characterized using solid state and solution NMR. The formation of oligomeric assemblies of TatA in the membrane, on the other hand, was investigated using in-plane neutron scattering of TatA reconstituted in aligned membranes. This way, we were able to both derive a detailed structural model of TatA, and to characterize pores formed by TatA.

The bacterial Twin-arginine translocation pathway is able to transport fully folded proteins across membranes. In B. subtilis it consists only of two components: TatCd, which serves as a receptor for the signal peptide, and the pore forming unit TatAd, which occurs in high stoichiometric excess. According to circular dichroism TatA contains a transmembrane segment, an amphiphilic helix, and an unstructured C-terminus [1]. Its detailed moelcular structure was resolved by solid-state NMR spectroscopy in oriented bilayers [2]. A striking pattern on the monomeric protein surface allowed us to assemble several units into protomers and into an open oligomeric pore. The stability of these complexes was supported by all-atom MD simulations and using structure-based modeling [3]. The observed interactions suggest that a novel motif for folding and self-assembly motif is present in this membrane-bound transport system, which allows reversible pore formation. Our comprehensive three-dimensional model thus reconciles for the first time TatA transport with a pore size of variable diameter, which can open and close by an energetically feasible mechanism.[1] Muller, S.D., A.A. De Angelis, T.H. Walther, S.L. Grage, C. Lange, S.J. Opella & A.S. Ulrich (2007) Biochim. Biophys. Acta 1768: 3071-3079[2] Walther, T.H., S.L. Grage, N. Roth & A.S. Ulrich (2010) J. Am. Chem. Soc., in press[3] Grage, S.L., T.H. Walther, M. Wolf, A. Vargiu, M.J. Klein, P. Ruggerone, W. Wenzel, A.S. Ulrich (2010) submitted∼

A mixture of 1,2-dimyristoyl- sn -glycero-3-phosphocholine (DMPC) with the short-chain detergent n-dodecylphosphocholine (DPC) is introduced here as a new membrane-mimetic bicelle system for solid-state NMR structure analysis of membrane proteins in oriented samples. Magnetically aligned DMPC/DPC bicelles are stable over a range of concentrations, with an optimum lipid ratio of q = 3:1, and they can be flipped with lanthanide ions. The advantage of DMPC/DPC over established bicelle systems lies in the possibility to use one and the same detergent for purification and NMR analysis of the membrane protein, without any need for detergent exchange. Furthermore, the same batch of protein can be studied in both micelles and bicelles, using liquid-state and solid-state NMR, respectively. The applicability of the DMPC/DPC bicelles is demonstrated here with the 15 N-labeled transmembrane protein TatA.

The transmembrane protein TatA is the pore forming unit of the twin-arginine translocase (Tat), which has the unique ability of transporting folded proteins across the cell membrane. This ATP-independent protein export pathway is a recently discovered alternative to the general secretory (Sec) system of bacteria. To obtain insight in the translocation mechanism, the structure and alignment in the membrane of the well-folded segments 2–45 of TatAd from Bacillus subtilis was studied here. Using solid-state NMR in bicelles containing anionic lipids, the topology and orientation of TatAd was determined in an environment mimicking the bacterial membrane. A wheel-like pattern, characteristic for a tilted transmembrane helix, was observed in 15N chemical shift /15N–1H dipolar coupling correlation NMR spectra. Analysis of this PISA wheel revealed a 14–16 residue long N-terminal membrane-spanning helix which is tilted by 17° with respect to the membrane normal. In addition, comparison of uniformly and selectively 15N-labeled TatA2–45 samples allowed determination of the helix polarity angle.