Biomedical subjects

R T Sauer

Publications and source records attributed to R T Sauer.

At least 55 records · Page 3Linked to original sources

PubMed1997

PDZ-like domains mediate binding specificity in the Clp/Hsp100 family of chaperones and protease regulatory subunits.

ClpX, a molecular chaperone and the regulatory subunit of the ClpXP protease, is shown to contain tandem modular domains that bind to the C-terminal sequences of target proteins in a manner that parallels functional specificity. Nuclear magnetic resonance studies show that these C-terminal sequences are displayed as disordered peptides on the surface of otherwise folded proteins. The ClpX substrate-binding domains are homologous to sequences in other Clp/Hsp100 proteins and are related more distantly to PDZ domains, which also mediate C-terminal specific protein-protein interactions. Conservation of these binding domains indicates that the mode of substrate recognition characterized here for ClpX will be a conserved feature among Clp/Hsp100 family members and a distinguishing characteristic between this chaperone family and the Hsp70 chaperones.

ATPases Associated with Diverse Cellular Activitie↗

PubMed1997

Formation of a denatured dimer limits the thermal stability of Arc repressor.

The thermal stability of the Arc repressor dimer normally increases with concentration because protein folding and subunit association are thermodynamically coupled. At Arc concentrations above 100 microM, however, thermal denaturation remains reversible and cooperative but tm does not continue to increase. In this concentration regime, thermally denatured Arc shows significantly reduced secondary structure and no evidence of a tightly packed core, but light scattering and fluorescence polarization studies indicate that the protein is dimeric. Higher order denatured oligomers are not observed and the stability of the non-native dimer is reduced by Arc mutations, indicating that non-native dimerization involves specific interactions between Arc subunits.

Circular Dichroism↗

PubMed1997

Engrailed (Gln50-->Lys) homeodomain-DNA complex at 1.9 A resolution: structural basis for enhanced affinity and altered specificity.

BACKGROUND: The homeodomain is one of the key DNA-binding motifs used in eukaryotic gene regulation, and homeodomain proteins play critical roles in development. The residue at position 50 of many homeodomains appears to determine the differential DNA-binding specificity, helping to distinguish among binding sites of the form TAATNN. However, the precise role(s) of residue 50 in the differential recognition of alternative sites has not been clear. None of the previously determined structures of homeodomain-DNA complexes has shown evidence for a stable hydrogen bond between residue 50 and a base, and there has been much discussion, based in part on NMR studies, about the potential importance of water-mediated contacts. This study was initiated to help clarify some of these issues. RESULTS: The crystal structure of a complex containing the engrailed Gln50-->Lys variant (QK50) with its optimal binding site TAATCC (versus TAATTA for the wild-type protein) has been determined at 1.9 A resolution. The overall structure of the QK50 variant is very similar to that of the wild-type complex, but the sidechain of Lys50 projects directly into the major groove and makes several hydrogen bonds to the O6 and N7 atoms of the guanines at base pairs 5 and 6. Lys50 also makes an additional water-mediated contact with the guanine at base pair 5 and has an alternative conformation that allows a hydrogen bond with the O4 of the thymine at base pair 4. CONCLUSIONS: The structural context provided by the folding and docking of the engrailed homeodomain allows Lys50 to make remarkably favorable contacts with the guanines at base pairs 5 and 6 of the binding site. Although many different residues occur at position 50 in different homeodomains, and although numerous position 50 variants have been constructed, the most striking examples of altered specificity usually involve introducing or removing a lysine sidechain from position 50. This high-resolution structure also confirms the critical role of Asn51 in homeodomain-DNA recognition and further clarifies the roles of water molecules near residues 50 and 51.

Animals↗

PubMed1996

Role of operator subsites in Arc repression.

By binding to adjacent subsites in its 21 base-pair operator, Arc represses transcription from two divergent promoters, Pant and Pmnt, in the immunity I operon of bacteriophage P22. Arc dimers bind to each subsite with nanomolar affinities and interact through protein-protein interactions to stabilize binding further. Here, we show that an Arc dimer bound to a single subsite reduces the rate of RNA polymerase open-complex formation and represses transcription from Pant and Pmnt promoter variants to varying degrees. Occupancy of the subsite proximal to the Pant-35 region results in significantly greater repression than occupancy of the- 10 proximal subsite. For repression of Pmnt, Arc bound at the- 10 proximal subsite is more effective than Arc bound at the- 35 proximal subsite. Because of the divergent orientations of the two promoters, the-35 proximal site in Pant is the same as the- 10 proximal site in Pmnt. Thus, in both cases, the same operator subsite is primarily responsible for repression of transcription initiation.

Base Sequence↗

PubMed1996

Equilibrium stability and sub-millisecond refolding of a designed single-chain Arc repressor.

Arc-L1-Arc is a single-chain variant of bacteriophage P22 Arc repressor in which a 15 residue linker joins the C-terminus of one subunit to the N-terminus of an otherwise identical subunit. Spectroscopic probes indicate that the native and denatured state of the single-chain protein are similar to those of the unlinked Arc dimer. In equilibrium experiments, Arc-L1-Arc denatures in a reaction without populated intermediate states as judged by the fits of the denaturation isotherms to a two-state model and by the coincidence of denaturation curves monitored by fluorescence and circular dichroism. Comparison of the equilibrium stabilities of Arc-L1-Arc and unlinked Arc gives an effective concentration of subunits in the denatured single-chain variant of 2.7 (+/- 0.7) mM. The kinetic refolding and unfolding reactions of Arc-L1-Arc also appear to proceed without populated intermediates. The rate constant for Arc-L1-Arc unfolding is about 2-fold faster than that of unlinked Arc, indicating that the linker mediates no significant contacts in the native structure that need to be broken to allow unfolding. As expected, the major effect of the linker occurs during the refolding reaction, where the effective subunit concentration calculated from the bimolecular and unimolecular refolding rate constants is 4.5 (+/- 1.8) mM. The transition states for the unfolding and refolding reactions of Arc-L1-Arc and wild-type Arc have similar solvent exposures as measured by the urea dependencies of the equilibrium and rate constants. In the absence of urea, the single-chain protein refolds very rapidly (kf approximately 10(4) s-1) in a reaction that is essentially complete in the sub-millisecond time regime.

Amino Acid Sequence↗

PubMed1996

Signal detection by the PhoQ sensor-transmitter. Characterization of the sensor domain and a response-impaired mutant that identifies ligand-binding determinants.

The PhoP-PhoQ two-component system is required for virulence and/or regulatory stress responses in enteric bacteria. The PhoQ protein responds to low concentrations of extracellular divalent cations by activating PhoP-mediated transcription of a set of genes. PhoQ is a member of a family of transmembrane proteins that contain a periplasmic sensor domain coupled to a cytoplasmic transmitter domain. Here, we describe the cloning, purification, and properties of a fragment of Escherichia coli PhoQ corresponding to the sensor domain. This fragment is monomeric in solution and has a circular dichroism spectrum indicative of a mixture of alphahelix and beta-sheet. Divalent cations do not affect the oligomeric state, circular dichroism spectrum, or fluorescence spectrum of the sensor domain but do stabilize this domain to denaturation in a fashion expected for a direct binding model. We have also constructed a mutant in which a cluster of acidic amino acids (EDDDDAE) in the sensor domain is replaced with conservative, uncharged residues (QNNNNAQ). The mutant sensor domain is indistinguishable from wild type in terms of oligomeric form and spectral properties but differs in being substantially more stable to urea denaturation, showing no additional stabilization in the presence of divalent cations, and showing little activation of PhoP-mediated transcription in response to divalent-cation starvation in vivo. These data are consistent with a model in which divalent cations bind to the acidic cluster of the wild-type sensor domain and stabilize a conformation that is inactive in signaling. Substituting uncharged residues for the acidic cluster appears to mimic the effect of divalent-cation binding by stabilizing the inactive conformation.

Amino Acid Sequence↗

PubMed1996

Dual regulation of open-complex formation and promoter clearance by Arc explains a novel repressor to activator switch.

In studies of variants of the P(ant) promoter of bacteriophage P22, the Arc protein was found not only to slow the rate at which RNA polymerase forms open complexes but also to accelerate the rate at which the enzyme clears the promoter. These dual activities permit Arc, bound at a single operator subsite, to act as an activator or as a repressor of different promoter variants. For example, Arc activates a P(ant) variant for which promoter clearance is rate limiting in the presence and absence of Arc but represses a closely related variant for which open-complex formation becomes rate limiting in the presence of Arc. The acceleration of promoter clearance by Arc requires occupancy of the operator subsite proximal to the -35 region and is diminished when Arc bears a mutation in Arg-23, a residue that makes a DNA-backbone contact in the operator complex.

DNA Footprinting↗

PubMed1996

Protein stabilization by removal of unsatisfied polar groups: computational approaches and experimental tests.

The role of polar and charged side chains in partially buried protein environments has been probed in a variant of Arc repressor (MYL) in which hydrophobic interactions between Met31, Tyr36, and Leu40 replace the wild-type salt-bridge interactions between Arg31, Glu36, and Arg40. In the absence of this salt-bridge triad, three additional side chains were identified by continuum electrostatic calculations as incurring larger desolvation penalties during folding than were recovered in favorable electrostatic interactions in the folded state. These side chains (Asn29, Ser44, and Glu48) were mutated singly and collectively to alanine in the MYL background, and the thermodynamic stabilities of the resulting mutant proteins were found to be increased by 0.1 to 1.3 kcal/mol of dimer. All of the mutants displayed cooperative thermal melts and appeared to have well-packed hydrophobic cores by near-UV circular dichroism spectroscopy, indicating that conformational specificity is maintained. The Arc variant (MYL-NA29/SA44/EA48) in which the entire six-residue polar network is replaced by nonpolar groups is 5.1 kcal/mol of dimer more stable than wild type, indicating that the strategy of replacing buried or partially buried charged and polar side chains with hydrophobic residues can lead to substantial stabilization.

Calorimetry↗

PubMed1996

Nonlinear free energy relationships in Arc repressor unfolding imply the existence of unstable, native-like folding intermediates.

Under strongly denaturing conditions, the logarithm of the rate constant for dissociation/unfolding of the wild-type Arc dimer varies in a nonlinear fashion with denaturant concentration. To assess the unfolding/dissociation behavior under conditions favoring the native structure, we mixed Arc variants labeled with fluorescence acceptor or donor groups and used energy transfer to monitor the increase in heterodimer with time. Under the conditions of this experiment, the rate at which the heterodimer concentration approaches its equilibrium value is determined by rate of dissociation and unfolding of the protein. Using this method and traditional denaturant-jump experiments, rate constants for unfolding/dissociation were determined over a wide range of stabilizing and destabilizing conditions. In each case examined, plots of log(ku) versus denaturant showed significant curvature under strongly denaturing conditions, even though other kinetic experiments indicates that the unfolding/dissociation reactions remain largely two-state. This curvature can be explained most readily by a series of unstable intermediates in the unfolding pathway, with denaturant-induced changes in the kinetic step that is rate-limiting. Alternatively, curvature might result from Hammond behavior in which the structure of the transition state becomes more native-like as the stability of native Arc decreases with increasing denaturant.

Amino Acid Sequence↗

PubMed1996

Barriers to protein folding: formation of buried polar interactions is a slow step in acquisition of structure.

In the MYL mutant of the Arc repressor dimer, sets of partially buried salt-bridge and hydrogen-bond interactions mediated by Arg-31, Glu-36, and Arg-40 in each subunit are replaced by hydrophobic interactions between Met-31, Tyr-36, and Leu-40. The MYL refolding/dimerization reaction differs from that of wild type in being 10- to 1250-fold faster, having an earlier transition state, and depending upon viscosity but not ionic strength. Formation of the wild-type salt bridges in a hydrophobic environment clearly imposes a kinetic barrier to folding, which can be lowered by high salt concentrations. The changes in the position of the transition state and viscosity dependence can be explained if denatured monomers interact to form a partially folded dimeric intermediate, which then continues folding to form the native dimer. The second step is postulated to be rate limiting for wild type. Replacing the salt bridge with hydrophobic interactions lowers this barrier for MYL. This makes the first kinetic barrier rate limiting for MYL refolding and creates a downhill free-energy landscape in which most molecules which reach the intermediate state continue to form native dimers.

Amino Acid Sequence↗

PubMed1996

Lac repressor at last.

Crystal and cocrystal structures of the LacI and PurR repressors reveal a novel use of hinge alpha helices which bind in the minor groove of the operator and mediate transmission of the allosteric signals that modulate DNA-binding activity.

Bacterial Proteins↗

PubMed1996

Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA.

Variants of lambda repressor and cytochrome b562 translated from messenger RNAs without stop codons were modified by carboxyl terminal addition of an ssrA-encoded peptide tag and subsequently degraded by carboxyl terminal-specific proteases present in both the cytoplasm and periplasm of Escherichia coli. The tag appears to be added to the carboxyl terminus of the nascent polypeptide chain by cotranslational switching of the ribosome from the damaged messenger RNA to ssrA RNA.

Alanine↗

PubMed1996

Sequence determinants of C-terminal substrate recognition by the Tsp protease.

Cytochrome b562 is not cleaved by the tail-specific protease Tsp in vitro or in the periplasm of Escherichia coli but becomes a good substrate when the C-terminal sequence WVAAA is added. Following randomization of the final three residue positions of this substrate, 54 different mutants with single residue substitutions were recovered. The steady-state expression levels of cytochrome variants bearing these mutant tails were similar in an E. coli strain deleted for the tsp gene but differed markedly in a strain containing Tsp. Wild-type cytochrome b562 and seven variants, displaying a range of intracellular expression levels, were purified. These proteins were found to have the same Tm values in thermal denaturation experiments but to be cleaved by Tsp at rates differing by as much as 30-fold. Overall, the rates of Tsp cleavage of these proteins in vitro correlate with their rates of cleavage in vivo as determined by pulse-chase experiments. These results indicate that the C-terminal sequence of the cytochrome-b562 variants is important in determining their proteolytic fate in the cell and show that this degradation is mediated predominantly by Tsp. There are different selectivity rules at each of the three C-terminal positions. The identity of the C-terminal residue of the substrate, where small, uncharged residues (Ala, Cys, Ser, Thr, Val) are preferred, is most important in determining the rate of substrate cleavage by Tsp. Non-polar residues are also preferred at the second and third positions, but larger and more hydrophobic side chains are also acceptable at these positions in good substrates.

Amino Acid Sequence↗

PubMed1996

Covalent attachment of Arc repressor subunits by a peptide linker enhances affinity for operator DNA.

By designing a recombinant gene containing tandem copies of the arc coding sequence with intervening DNA encoding the linker sequence GGGSGGGTGGGSGGG, the two subunits of the P22 Are repressor dimer have been covalently linked to form a single-chain protein called Arc-L1-Arc. The 15-residue linker joins the C-terminus of one monomer to the N-terminus of the second, a distance of approximately 45 A in the Arc-operator cocrystal structure. Arc-L1-Arc is expressed at high levels in Escherichia coli, with no evidence of degradation or proteolytic clipping of the linker, and is more active than wild-type Arc in repression assays. The purified Arc-L1-Arc protein has the molecular weight expected for the designed protein and unfolds cooperatively, reversibly, and with no concentration dependence in thermal-denaturation studies. Arc-L1-Arc protects operator DNA in a manner indistinguishable from that of wild-type Arc in DNase I and copper-phenanthroline footprinting studies, but the covalent attachment of the two monomers results in enhanced affinity for operator DNA. Arc-L1-Arc binds operator DNA half-maximally at a concentration of 1.7 pM, compared with the wild-type value of 185 pM, and also binds DNA fragments containing the left or right operator half-sites more tightly than wild type. Because wild-type Arc is monomeric at sub-nanomolar concentrations and must dimerize before binding to the operator, it was anticipated that Arc-L1-Arc would exhibit a lower half-maximal binding concentration. However, even when the change from a monomeric to a dimeric species is taken into account, the affinity of Arc-L1-Arc for operator and half-operator DNA is greater than the wild-type affinity. This tighter binding appears to result from slower dissociation, as Arc-L1-Arc DNA complexes with full or half-site operators dissociate at rates 5-10 times slower than the corresponding Arc--DNA complexes. Hence, the activity of the designed Arc-L1-Arc protein is substantially increased relative to wild-type Arc in a variety of assays.

Amino Acid Sequence↗

PubMed1996

Sequence space, folding and protein design.

Protein design efforts are beginning to yield molecules with many of the properties of natural proteins. Such experiments are informed by and contribute to our understanding of the sequence determinants of protein folding and stability. The most important design elements seem to be the proper placement of hydrophobic residues along the polypeptide chain and the ability of these residues to form a well packed core. Buried polar interactions, turn and capping motifs and secondary structural propensities also contribute, although probably to a lesser extent.

Amino Acid Sequence↗

PubMed1996

Protein folding from a combinatorial perspective.

Combinatorial mutagenesis experiments show the existence of many different solutions to the problem of complementary packing of non-polar sidechains in the protein core. They suggest that a significant amount of structural information is carried by the simple pattern of polar and non-polar residues along the polypeptide chain, indicate that the formation of buried polar interactions may be a fundamentally slow step in protein folding and show that proteins with many native properties occur at reasonable frequencies in random sequence libraries.

DNA-Binding Proteins↗

PubMed1996

Sequence determinants of folding and stability for the P22 Arc repressor dimer.

The Arc repressor is a small, homodimeric protein. Studies of mutant proteins show that the side chains that form the hydrophobic core are the most important determinants of structure. A variety of hydrogen bonds and salt bridges also contribute to stabilization of the native structure, but these can often be replaced by hydrophobic interactions. The transition state for folding/unfolding is dimeric and contains a large amount of buried hydrophobic surface, but the beta-sheet of native Arc is not formed. Moreover, relatively little side chain information appears to be used in the transition state, suggesting that tight packing of the hydrophobic core and optimization of hydrogen-bond geometry are events that occur later in folding.

Amino Acid Sequence↗

PubMed1996

The DegP and DegQ periplasmic endoproteases of Escherichia coli: specificity for cleavage sites and substrate conformation.

DegP and DegQ are homologous endoproteases found in the periplasmic compartment of Escherichia coli. The studies presented here suggest that DegP and DegQ have very similar substrate specificities and cleave substrates which are transiently or globally denatured. Model substrates were cleaved at discrete Val/Xaa or Ile/Xaa sites, suggesting that aliphatic, beta-branched residues, which are typically buried in the hydrophobic core of most proteins, are important determinants of cleavage specificity. Indeed, the peptide bonds cleaved in the model substrates are generally inaccessible in the native three-dimensional structures. In addition, a chimeric fusion protein, which is a DegP substrate in vivo, is degraded in vitro only after reduction of its intramolecular disulfide bonds. Taken together, these findings suggest that DegP and DegQ may degrade transiently denatured proteins, unfolded proteins which accumulate in the periplasm following heat shock or other stress conditions, and/or newly secreted proteins prior to folding and disulfide bond formation. Cross-linking studies indicate that both DegP and DegQ form dodecamers in solution and thus are similar to many other intracellular proteases which form large oligomeric complexes.

Amino Acid Sequence↗