Non-functional Conformation Research Articles

Structure-based Drug Design Targeting an Inactive RNA Conformation: Exploiting the Flexibility of HIV-1 TAR RNA

The targeting of RNA for the design of novel anti-viral compounds represents an area of vast potential. We have used NMR and computational methods to model the interaction of a series of synthetic inhibitors of the in vitro RNA binding activities of a peptide derived from the transcriptional activator protein, Tat, from human immunodeficiency virus type 1. Inhibition has been measured through the monitering of fluorescence resonance energy transfer between fluorescently labeled peptide and RNA components. A series of compounds containing a bi-aryl heterocycle as one of the three substituents on a benzylic scaffold, induce a novel, inactive TAR conformation by stacking between base-pairs at the site of a three-base bulge within TAR. The development of this series resulted in an enhancement in potency (with K i <100 nM in an in vitro assay) and the removal of problematic guanidinium moieties. Ligands from this series can act as inhibitors of Tat-induced transcription in a cell-free system. This study validates the drug design strategy of using a ligand to target the RNA receptor in a non-functional conformation.

Regulation of Initiator Caspase Activity

Apoptosis, or cell suicide, depends upon activation of the caspases, a family of cysteine proteases. Caspases, which exist as inactive proenzymes, are activated in a two-stage proteolytic cascade. Initiator caspases are activated after recruitment to ligand-bound death receptors (the extrinsic pathway) or to the apoptosome (the intrinsic pathway). The initiator caspases cleave and activate executioner caspases, which cleave various cellular constituents. Caspase-9, an initiator caspase in the intrinsic pathway, forms a homodimer in which one monomer assumes a catalytically active conformation and the other assumes an inactive conformation. In contrast to regulation of the executioner caspases, regulation of caspase-9 activity depends upon dimerization rather than upon proteolysis. Shiozaki et al. investigated the mechanism by which the X-linked inhibitor of apoptosis (XIAP) protein regulates caspase-9 and discovered that this depended upon XIAP inhibition of caspase-9 homodimerization. Structural analysis of a complex formed by regions of caspase-9 and the third baculoviral inhibitory domain (BIR3, the caspase-9 inhibiting region) of XIAP indicated that they formed a heterodimer. BIR3 interacted with the region of caspase-9 that normally mediates homodimerization and trapped the active site in a nonfunctional conformation. Mutational analysis confirmed that an obligatory monomer was catalytically inactive and that a sequence from one member of a caspase-9 dimer was required for activity of the second. Thus, XIAP both maintains one monomer of caspase-9 in an inactive form and prevents assembly of the functional dimeric protease. In related research, Boatright et al. and Donepudi et al. demonstrated that catalytic activity of caspase-8, an initiator caspase in the extrinsic pathway, also depended upon dimerization rather than upon proteolysis. E. N. Shiozaki, J. Chai, D. L. Rigotti, S. J. Riedi, P. Li, S. M. Srinivasula, E. S. Alnemri, R. Fairman, Y. Shi, Mechanism of XIAP-mediated inhibition of caspase-9. Molecular Cell 11 , 519-527 (2003). [Online Journal] K. M. Boatright, M. Renatus, F. L. Scott, S. Sperandio, H. Shin, I. M. Pederson, J,-E. Ricci, W. A. Edris, D. P. Sutherlin, D. R. Green, G. S. Salvesen, A unified model for apical caspase activation. Molecular Cell 11 , 520-541 (2003). [Online Journal] M. Donepudi, A. M. Sweeney, C. Briand, M. G. Grütter, Insights into the regulatory mechanism for caspase-8 activation. Molecular Cell 11 , 543-549 (2003). [Online Journal]

Regulation of Initiator Caspase Activity

Apoptosis, or cell suicide, depends upon activation of the caspases, a family of cysteine proteases. Caspases, which exist as inactive proenzymes, are activated in a two-stage proteolytic cascade. Initiator caspases are activated after recruitment to ligand-bound death receptors (the extrinsic pathway) or to the apoptosome (the intrinsic pathway). The initiator caspases cleave and activate executioner caspases, which cleave various cellular constituents. Caspase-9, an initiator caspase in the intrinsic pathway, forms a homodimer in which one monomer assumes a catalytically active conformation and the other assumes an inactive conformation. In contrast to regulation of the executioner caspases, regulation of caspase-9 activity depends upon dimerization rather than upon proteolysis. Shiozaki et al. investigated the mechanism by which the X-linked inhibitor of apoptosis (XIAP) protein regulates caspase-9 and discovered that this depended upon XIAP inhibition of caspase-9 homodimerization. Structural analysis of a complex formed by regions of caspase-9 and the third baculoviral inhibitory domain (BIR3, the caspase-9 inhibiting region) of XIAP indicated that they formed a heterodimer. BIR3 interacted with the region of caspase-9 that normally mediates homodimerization and trapped the active site in a nonfunctional conformation. Mutational analysis confirmed that an obligatory monomer was catalytically inactive and that a sequence from one member of a caspase-9 dimer was required for activity of the second. Thus, XIAP both maintains one monomer of caspase-9 in an inactive form and prevents assembly of the functional dimeric protease. In related research, Boatright et al. and Donepudi et al. demonstrated that catalytic activity of caspase-8, an initiator caspase in the extrinsic pathway, also depended upon dimerization rather than upon proteolysis.E. N. Shiozaki, J. Chai, D. L. Rigotti, S. J. Riedi, P. Li, S. M. Srinivasula, E. S. Alnemri, R. Fairman, Y. Shi, Mechanism of XIAP-mediated inhibition of caspase-9. Molecular Cell 11, 519-527 (2003). [Online Journal]K. M. Boatright, M. Renatus, F. L. Scott, S. Sperandio, H. Shin, I. M. Pederson, J,-E. Ricci, W. A. Edris, D. P. Sutherlin, D. R. Green, G. S. Salvesen, A unified model for apical caspase activation. Molecular Cell 11, 520-541 (2003). [Online Journal]M. Donepudi, A. M. Sweeney, C. Briand, M. G. Grütter, Insights into the regulatory mechanism for caspase-8 activation. Molecular Cell 11, 543-549 (2003). [Online Journal]

Native-like interactions favored in the unfolded bovine pancreatic trypsin inhibitor have different roles in folding.

Folding kinetics of a series of bovine pancreatic trypsin inhibitor (BPTI) variants with similar stabilities and structures have been measured. All are strongly destabilized relative to WT. In Y21A, F22A, Y23A, G37A, and F45A, the three native disulfide bonds are retained. In RM(14-38), Cys14 and Cys38 thiols are methylated while C30-C51 and C5-C55 disulfides remain intact. At pH 2 and 20 degrees C, relaxation rate constants of the major kinetic phase range from approximately 10 ms to 0.71 s in the absence of denaturant. All mutants except G37A exhibit standard two-state behavior. Y21A, F22A, and Y23A fold much more slowly than other mutants. The experiments were designed to test the hypothesis that native-like structure detected in the unfolded BPTI is important in folding. Two native-like contacts are implied by NOEs in reduced and unfolded BPTI, between residues Tyr23 and Ala25, and between Gly37 NH and the Tyr35 ring. The results support an earlier hypothesis that formation of the central beta-hairpin, monitored by a local native interaction between Tyr23 and Ala25, is crucial to initiation of BPTI folding. The second native-like contact is important, not in folding initiation, but in preventing a kinetic trap later in the process. Evidence for this comes from mutant G37A, which behaves very differently from the others in displaying a phenomenon called rollover. G37A is, to our knowledge, the first reported case in which a single-site replacement causes rollover, while the wild type and all other known mutants of the same protein show typical two-state chevron plots. The best explanation is that the G37A mutation introduces a kinetic trap of the type described by Chan and Dill [(1998) Proteins 30, 2-33]. In native BPTI, there is an unusual polar interaction between the ring of Tyr35 and the backbone NH of Gly37. Our results suggest that the NH-aromatic interaction between residues 37 and 35 is important throughout folding in stabilizing native-like loop conformations and in preventing the flexible loops from being trapped in nonfunctional conformations during later stages of folding.

Truncated aspartate aminotransferase from alkalophilic Bacillus circulans with deletion of N-terminal 32 amino acids is a non-functional monomer in a partially structured state

Aspartate aminotransferase (AspAT) from alkalophilic Bacillus circulans contains an additional N-terminal sequence of 32 amino acid residues that are absent in all other AspATs from different sources. Modeling suggested that this sequence forms two alpha-helical segments which establish a continuous network of interactions on the surface of the molecule. In the present study, we studied the role of the N-terminal sequence in folding and stability of AspAT by applying the scanning calorimetry, and CD and fluorescence spectroscopies to the native and truncated enzymes. Truncated AspAT (Delta2alpha mutant) devoid of N-terminal residues cannot provide sufficient potential of quaternary intersubunit and subunit-cofactor interactions, which results in a monomeric non-functional conformation. However, the residual tertiary interactions in the Delta2alpha mutant are sufficient to: i) provide stability of a residual structure over a wide pH range; ii) confer moderate cooperativity of the denaturant-induced transition while only low cooperativity of the thermal transition, and iii) maintain the hydrophobic core of a part of the structure which prevents aromatic fluorophores from quenching by water. Furthermore, the present study provides evidence that AspAT from the alkalophilic bacterium follows unfolding pathway comprising a stable non-functional intermediate, in contrast to a two-state mechanism of the thermophilic AspAT from Sulfolobus solfataricus.

Mot protein assembly into the bacterial flagellum: a model based on mutational analysis of the motB gene

The 308 residue MotB protein anchors the stator complex of the Escherichia coli flagellar motor to the peptidoglycan of the cell wall. Together with MotA, it comprises the transmembrane channel that delivers protons to the motor. At the outset of the mutational analysis of MotB described here, we found that the non-motile phenotype of a Δ motAB strain was rescued better by a p motA + B + plasmid than the non-motile phenotype of a Δ motB strain was rescued by a p motB + plasmid. Transcription in each case was from the inducible tac promoter but relied on the native ribosome-binding site (RBS). This result confirms that translational coupling to motA is important for normal translation of the motB mRNA, since overproduction of MotA in trans did not improve complementation by p motB. However, introduction of an optimized RBS into p motB (to generate p motB o) did. To dissect the function of the periplasmic domain of MotB, site-directed mutagenesis was used to replace Gln, Ser, and Tyr codons scattered throughout motB with amber (UAG) codons. Plasmid-borne motB am genes were introduced into sup o, supE, and supF strains to see what motility defects were imposed by particular amber mutations and whether the defects could be suppressed by amber-suppressor tRNAs inserting the native or heterologous amino acids. Amber mutations at codon 268 or earlier in p motB, and at codon 261 or earlier in p motB o or p motAB, eliminated motility. Thus, in agreement with the deletion analysis of motB by another laboratory, we conclude that the portion of MotB carboxyl-terminal to its peptidoglycan-binding motif (residues 161 to 264) is not essential. In strains containing supE or supF alleles, motility defects associated with motB am mutations were suppressed weakly, if at all, in p motB. In contrast, motility defects conferred by most motB am mutations in p motB o or p motAB could be suppressed to a significant extent. However, the S18 am, Q100 am, Q112 am, Q124 am, Y201 am, and Y208 am mutations were still suppressed extremely poorly. Full-length MotB was present at very low levels in suppressor strains containing the first four mutations, but Y201 am and Y208 am were suppressed efficiently at the translational level. We suggest that a translational pause by suppressor tRNAs reading UAG at these two positions may divert the nascent polypeptide into an alternative folding pathway that traps MotB in a non-functional conformation. We further propose that MotA and MotB form a stable pre-assembly complex in the membrane. In this complex, MotB exists in a form that cannot associate with peptidoglycan and blocks the proton-conducting channel. Opening of the channel and attachment to the cell wall may occur when the complex collides with a flagellar basal body and MotA makes specific contacts with the C ring and/or the MS ring.

Distinguishing surface effects of calcium ion from pore-occupancy effects in Na+ channels.

The effects of calcium ion on the Na+ activation gate were studied in squid giant axons. Saxitoxin (STX) was used to block ion entry into Na+ channels without hindering access to the membrane surface, making it possible to distinguish surface effects of calcium from pore-occupancy effects. In the presence of STX, gating kinetics were measured from gating current (Ig). The kinetic effects of external calcium concentration changes were small when STX was present. In the absence of STX, lowering the calcium concentration (from 100 to 10 mM) slowed the closing of Na+ channels (measured from INa tails) by more than a factor of 2. Surprisingly, the voltage sensitivity of closing kinetics changed with calcium concentration, and it was modified by STX. Voltage sensitivity apparently depends in part on the ability of calcium to enter and block the channels as voltage is driven negative. In external medium with no added calcium, INa tail current initially increases in amplitude severalfold with the relief of calcium block, then progressively slows and gets smaller, as calcium diffuses out of the layers investing the axon. INa tails seen just before the current disappears suggest that closing in the absence of channel block is very slow or does not occur. INa amplitude and kinetics are completely restored when calcium is returned. The results strongly suggest that calcium occupancy is a requirement for channel closing and that nonoccupied channels fold reversibly into a nonfunctional conformation.

The transcriptional activity of a muscle-specific promoter depends critically on the structure of the TATA element and its binding protein

We have previously characterized the proximal promoter of the mouse IIB myosin heavy chain (MyHC) gene, which is expressed only in fast-contracting glycolytic skeletal muscle fibers. We show here that the substitution into this promoter of a non-canonical TATA sequence from the IgH gene results in inactivity in muscle cells, even though TATA-binding protein (TBP) can bind strongly to this mutated promoter. Chemical footprinting data show, however, that TBP makes different DNA contacts on this heterologous TATA sequence. The inactivity of such a non-canonical TATA motif in the IIB promoter context appears to be caused by a non-functional conformation of the bound TBP-DNA complex that is incapable of sustaining transcription. The conclusions imply that the precise sequence of the promoter TATA motif needs to be matched with the specific functional class of upstream activator proteins present in a given cell type in order for the gene to be transcriptionally active.