Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains.

Bak and Bax mediate apoptotic cell death by oligomerizing and forming a pore in the mitochondrial outer membrane. Both proteins anchor to the outer membrane via a C-terminal transmembrane domain, although its topology within the apoptotic pore is not known. Cysteine-scanning mutagenesis and hydrophilic labeling confirmed that in healthy mitochondria the Bak α9 segment traverses the outer membrane, with 11 central residues shielded from labeling. After pore formation those residues remained shielded, indicating that α9 does not line a pore. Bak (and Bax) activation allowed linkage of α9 to neighboring α9 segments, identifying an α9:α9 interface in Bak (and Bax) oligomers. Although the linkage pattern along α9 indicated a preferred packing surface, there was no evidence of a dimerization motif. Rather, the interface was invoked in part by Bak conformation change and in part by BH3:groove dimerization. The α9:α9 interaction may constitute a secondary interface in Bak oligomers, as it could link BH3:groove dimers to high-order oligomers. Moreover, as high-order oligomers were generated when α9:α9 linkage in the membrane was combined with α6:α6 linkage on the membrane surface, the α6-α9 region in oligomerized Bak is flexible. These findings provide the first view of Bak carboxy terminus (C terminus) membrane topology within the apoptotic pore.

Structure of the cytoplasmic beta subunit-T1 assembly of voltage-dependent K+ channels.

The structure of the cytoplasmic assembly of voltage-dependent K+ channels was solved by x-ray crystallography at 2.1 angstrom resolution. The assembly includes the cytoplasmic (T1) domain of the integral membrane alpha subunit together with the oxidoreductase beta subunit in a fourfold symmetric T1(4)beta4 complex. An electrophysiological assay showed that this complex is oriented with four T1 domains facing the transmembrane pore and four beta subunits facing the cytoplasm. The transmembrane pore communicates with the cytoplasm through lateral, negatively charged openings above the T1(4)beta4 complex. The inactivation peptides of voltage-dependent K(+) channels reach their site of action by entering these openings.

Structure of a voltage-dependent K+ channel beta subunit.

The integral membrane subunits of many voltage-dependent potassium channels are associated with an additional protein known as the beta subunit. One function of beta subunits is to modify K+ channel gating. We have determined the structure of the conserved core of mammalian beta subunits by X-ray crystallography at 2.8 A resolution. Like the integral membrane component of K+ channels, beta subunits form a four-fold symmetric structure. Each subunit is an oxidoreductase enzyme complete with a nicotinamide co-factor in its active site. Several structural features of the enzyme active site, including its location with respect to the four-fold axis, imply that it may interact directly or indirectly with the K+ channel's voltage sensor. This structure suggests a mechanism for coupling membrane electrical excitability directly to chemistry of the cell.

The structure of the potassium channel: molecular basis of K+ conduction and selectivity.

The potassium channel from Streptomyces lividans is an integral membrane protein with sequence similarity to all known K+ channels, particularly in the pore region. X-ray analysis with data to 3.2 angstroms reveals that four identical subunits create an inverted teepee, or cone, cradling the selectivity filter of the pore in its outer end. The narrow selectivity filter is only 12 angstroms long, whereas the remainder of the pore is wider and lined with hydrophobic amino acids. A large water-filled cavity and helix dipoles are positioned so as to overcome electrostatic destabilization of an ion in the pore at the center of the bilayer. Main chain carbonyl oxygen atoms from the K+ channel signature sequence line the selectivity filter, which is held open by structural constraints to coordinate K+ ions but not smaller Na+ ions. The selectivity filter contains two K+ ions about 7.5 angstroms apart. This configuration promotes ion conduction by exploiting electrostatic repulsive forces to overcome attractive forces between K+ ions and the selectivity filter. The architecture of the pore establishes the physical principles underlying selective K+ conduction.

Structure of the guanine nucleotide exchange factor Sec7 domain of human arno and analysis of the interaction with ARF GTPase.

Sec7-related guanine nucleotide exchange factors (GEFs) initiate vesicle budding from the Golgi membrane surface by converting the GTPase ARF to a GTP-bound, membrane-associated form. Here we report the crystal structure of the catalytic Sec7 homology domain of Arno, a human GEF for ARF1, determined at 2.2 angstroms resolution. The Sec7 domain is an elongated, all-helical protein with a distinctive hydrophobic groove that is phylogenetically conserved. Structure-based mutagenesis identifies the groove and an adjacent conserved loop as the ARF-interacting surface. The sites of Sec7 domain interaction on ARF1 have subsequently been mapped, by protein footprinting experiments, to the switch 1 and switch 2 GTPase regions, leading to a model for the interaction between ARF GTPases and Sec7 domain exchange factors.

The influence of the proliferating cell nuclear antigen-interacting domain of p21(CIP1) on DNA synthesis catalyzed by the human and Saccharomyces cerevisiae polymerase delta holoenzymes.

In eukaryotes, processive DNA synthesis catalyzed by DNA polymerases delta and epsilon (pol delta and epsilon) requires the proliferating cell nuclear antigen (PCNA). It has recently been shown that in humans (h), the PCNA function, required for both DNA replication and nucleotide excision repair, can be inactivated by p21(CIP1) due to a specific interaction between hPCNA and the carboxyl terminus of p21(CIP1). In this report, we show that Saccharomyces cerevisiae (S. cerevisiae) PCNA-dependent pol delta-catalyzed DNA synthesis was inhibited less efficiently than the human system by the intact p21(CIP1) protein and was unaffected by the p21(CIP1) carboxyl-terminal peptide (codons 139-160). This species-specific response of PCNA to p21(CIP1)-mediated inhibition of DNA synthesis results from a marked difference in the ability of h and S. cerevisiae PCNA to interact with p21(CIP1). As shown by binding studies using the surface plasmon resonance technique, hPCNA binds both full-length p21(CIP1) and the p21(CIP1) peptide-(139-160) stoichiometrically with a similar affinity (KD approximately 2.5 nM) while S. cerevisiae PCNA binds p21(CIP1) with approximately 10-fold less affinity and does not interact with the p21(CIP1) peptide-(139-160).

Structure of the C-terminal region of p21(WAF1/CIP1) complexed with human PCNA.

The crystal structure of the human DNA polymerase delta processivity factor PCNA (proliferating cell nuclear antigen) complexed with a 22 residue peptide derived from the C-terminus of the cell-cycle checkpoint protein p21(WAF1/CIP1) has been determined at 2.6 angstrom resolution. p21 binds to PCNA in a 1:1 stoichiometry with an extensive array of interactions that include the formation of a beta sheet with the interdomain connector loop of PCNA. An intact trimeric ring is maintained in the structure of the p21-PCNA complex, with a central hole available for DNA interaction. The ability of p21 to inhibit the action of PCNA is therefore likely to be due to its masking of elements on PCNA that are required for the binding of other components of the polymerase assembly.

Crystal structure of a suicidal DNA repair protein: the Ada O6-methylguanine-DNA methyltransferase from E. coli.

The mutagenic and carcinogenic effects of simple alkylating agents are mainly due to methylation at the O6 position of guanine in DNA. O6-methylguanine directs the incorporation of either thymine or cytosine without blocking DNA replication, resulting in GC to AT transition mutations. In prokaryotic and eukaryotic cells antimutagenic repair is effected by direct reversal of this DNA damage. A suicidal methyltransferase repair protein removes the methyl group from DNA to one of its own cysteine residues. The resulting self-methylation of the active site cysteine renders the protein inactive. Here we report the X-ray structure of the 19 kDa C-terminal domain of the Escherichia coli ada gene product, the prototype of these suicidal methyltransferases. In the crystal structure the active site cysteine is buried. We propose a model for the significant conformational change that the protein must undergo in order to bind DNA and effect methyl transfer.

Structure of a dideoxynucleoside active against the HIV (AIDS) virus.

N4-Benzoyl-2',3'-dideoxycytidine, C16H17-N3O4.2H2O, M(r) = 351.4, monoclinic, P2(1), a = 4.691 (1), b = 14.448 (2), c = 12.924 (3) A, beta = 97.63 (1) degree, V = 868.2 (1) A3, Dm(flotation) = 1.34 (1), Dx = 1.344 Mg m-3, Z = 2, F(000) = 372, lambda = 1.5418 A, mu(Cu K alpha) = 0.65 mm-1, T = 292 (1) K, final R = 0.038 for 1437 observed data. The glycosidic torsion angle C(6)--N(1)--C(1')--O(4') is 20.6 (5) degrees and the pucker of the furanose ring is C(3') endo. Free rotation about the exocyclic C(4')--C(5') bond allows the hydroxymethyl substituent to adopt two orientations, trans and gauche, the latter resulting in a short contact, H(6)...O(5") of 2.21 (4) A, indicative of a relatively strong C--H...O intramolecular hydrogen-bonding interaction.