Structure and function in rhodopsin: Rhodopsin mutants with a neutral amino acid at E134 have a partially activated conformation in the dark state*

The Glu-134–Arg-135 residues in rhodopsin, located near the cytoplasmic end of the C helix, are involved in G protein binding, or activation, or both. Furthermore, the charge-neutralizing mutation Glu-134 to Gln-134 produces hyperactivity in the activated state and produces constitutive activity in opsin. The Glu/Asp-Arg charge pair is highly conserved in equivalent positions in other G protein-coupled receptors. To investigate the structural consequences of charge-neutralizing mutations at Glu-134 and Arg-135 in rhodopsin, single spin-labeled side chains were introduced at sites in the cytoplasmic domains of helices C (140), E (227), F (250), or G (316) to serve as “molecular sensors” of the local helix bundle conformation. In each of the spin-labeled rhodopsins, a Gln substitution was introduced at either Glu-134 or Arg-135, and the electron paramagnetic resonance spectrum of the spin label was used to monitor the structural response of the helix bundle. The results indicate that a Gln substitution at Glu-134 induces a photoactivated conformation around helices C and G even in the dark state, an observation of potential relevance to the hyperactivity and constitutive activity of the mutant. In contrast, little change is induced in helix F, which has been shown to undergo a dominant motion upon photoactivation. This result implies that the multiple helix motions accompanying photoactivation are not strongly coupled and can be induced to take place independently. Gln substitution at Arg-135 produces only minor structural changes in the dark- or light-activated conformation, suggesting that this residue is not a determinant of structure in the regions investigated, although it may be functionally important.

The anticoagulant activation of antithrombin by heparin

Antithrombin, a plasma serpin, is relatively inactive as an inhibitor of the coagulation proteases until it binds to the heparan side chains that line the microvasculature. The binding specifically occurs to a core pentasaccharide present both in the heparans and in their therapeutic derivative heparin. The accompanying conformational change of antithrombin is revealed in a 2.9-Å structure of a dimer of latent and active antithrombins, each in complex with the high-affinity pentasaccharide. Inhibitory activation results from a shift in the main sheet of the molecule from a partially six-stranded to a five-stranded form, with extrusion of the reactive center loop to give a more exposed orientation. There is a tilting and elongation of helix D with the formation of a 2-turn helix P between the C and D helices. Concomitant conformational changes at the heparin binding site explain both the initial tight binding of antithrombin to the heparans and the subsequent release of the antithrombin–protease complex into the circulation. The pentasaccharide binds by hydrogen bonding of its sulfates and carboxylates to Arg-129 and Lys-125 in the D-helix, to Arg-46 and Arg-47 in the A-helix, to Lys-114 and Glu-113 in the P-helix, and to Lys-11 and Arg-13 in a cleft formed by the amino terminus. This clear definition of the binding site will provide a structural basis for developing heparin analogues that are more specific toward their intended target antithrombin and therefore less likely to exhibit side effects.

The Putative “Switch 2” Domain of the Ras-related GTPase, Rab1B, Plays an Essential Role in the Interaction with Rab Escort Protein

Posttranslational modification of Rab proteins by geranylgeranyltransferase type II requires that they first bind to Rab escort protein (REP). Following prenylation, REP is postulated to accompany the modified GTPase to its specific target membrane. REP binds preferentially to Rab proteins that are in the GDP state, but the specific structural domains involved in this interaction have not been defined. In p21 Ras, the α2 helix of the Switch 2 domain undergoes a major conformational change upon GTP hydrolysis. Therefore, we hypothesized that the corresponding region in Rab1B might play a key role in the interaction with REP. Introduction of amino acid substitutions (I73N, Y78D, and A81D) into the putative α2 helix of Myc-tagged Rab1B prevented prenylation of the recombinant protein in cell-free assays, whereas mutations in the α3 and α4 helices did not. Additionally, upon transient expression in transfected HEK-293 cells, the Myc-Rab1B α2 helix mutants were not efficiently prenylated as determined by incorporation of [3H]mevalonate. Metabolic labeling studies using [32P]orthophosphate indicated that the poor prenylation of the Rab1B α2 helix mutants was not directly correlated with major disruptions in guanine nucleotide binding or intrinsic GTPase activity. Finally, gel filtration analysis of cytosolic fractions from 293 cells that were coexpressing T7 epitope-tagged REP with various Myc-Rab1B constructs revealed that mutations in the α2 helix of Rab1B prevented the association of nascent (i.e., nonprenylated) Rab1B with REP. These data indicate that the Switch 2 domain of Rab1B is a key structural determinant for REP interaction and that nucleotide-dependent conformational changes in this region are largely responsible for the selective interaction of REP with the GDP-bound form of the Rab substrate.

Reprogramming the Cell Cycle for Endoreduplication in Rodent Trophoblast Cells

Differentiation of trophoblast giant cells in the rodent placenta is accompanied by exit from the mitotic cell cycle and onset of endoreduplication. Commitment to giant cell differentiation is under developmental control, involving down-regulation of Id1 and Id2, concomitant with up-regulation of the basic helix-loop-helix factor Hxt and acquisition of increased adhesiveness. Endoreduplication disrupts the alternation of DNA synthesis and mitosis that maintains euploid DNA content during proliferation. To determine how the mammalian endocycle is regulated, we examined the expression of the cyclins and cyclin-dependent kinases during the transition from replication to endoreduplication in the Rcho-1 rat choriocarcinoma cell line. We cultured these cells under conditions that gave relatively synchronous endoreduplication. This allowed us to study the events that occur during the transition from the mitotic cycle to the first endocycle. With giant cell differentiation, the cells switched cyclin D isoform expression from D3 to D1 and altered several checkpoint functions, acquiring a relative insensitivity to DNA-damaging agents and a coincident serum independence. The initiation of S phase during endocycles appeared to involve cycles of synthesis of cyclins E and A, and termination of S was associated with abrupt loss of cyclin A and E. Both cyclins were absent from gap phase cells, suggesting that their degradation may be necessary to allow reinitiation of the endocycle. The arrest of the mitotic cycle at the onset of endoreduplication was associated with a failure to assemble cyclin B/p34cdk1 complexes during the first endocycle. In subsequent endocycles, cyclin B expression was suppressed. Together these data suggest several points at which cell cycle regulation could be targeted to shift cells from a mitotic to an endoreduplicative cycle.

Altered recognition mutants of the response regulator PhoB: A new genetic strategy for studying protein–protein interactions

Two-component regulatory systems require highly specific interactions between histidine kinase (transmitter) and response regulator (receiver) proteins. We have developed a novel genetic strategy that is based on tightly regulated synthesis of a given protein to identify domains and residues of an interacting protein that are critical for interactions between them. Using a reporter strain synthesizing the nonpartner kinase VanS under tight arabinose control and carrying a promoter-lacZ fusion activated by phospho-PhoB, we isolated altered recognition (AR) mutants of PhoB showing enhanced activation (phosphorylation) by VanS as arabinose-dependent Lac+ mutants. Changes in the PhoBAR mutants cluster in a “patch” near the proposed helix 4 of PhoB based on the CheY crystal structure (a homolog of the PhoB receiver domain) providing further evidence that helix 4 lies in the kinase-regulator interface. Based on the CheY structure, one mutant has an additional change in a region that may propagate a conformational change to helix 4. The overall genetic strategy described here may also be useful for studying interactions of other components of the vancomycin resistance and Pi signal transduction pathways, other two-component regulatory systems, and other interacting proteins. Conditionally replicative oriRR6Kγ attP “genome targeting” suicide plasmids carrying mutagenized phoB coding regions were integrated into the chromosome of a reporter strain to create mutant libraries; plasmids encoding mutant PhoB proteins were subsequently retrieved by P1-Int-Xis cloning. Finally, the use of similar genome targeting plasmids and P1-Int-Xis cloning should be generally useful for constructing genomic libraries from a wide array of organisms.

Staphylococcal protein A: Unfolding pathways, unfolded states, and differences between the B and E domains

We present multiple native and denaturation simulations of the B and E domains of the three-helix bundle protein A, totaling 60 ns. The C-terminal helix (H3) consistently denatures later than either of the other two helices and contains residual helical structure in the denatured state. These results are consistent with experiments suggesting that the isolated H3 fragment is more stable than H1 and H2 and that H3 forms early in folding. Interestingly, the denatured state of the B domain is much more compact than that of the E domain. This sequence-dependent effect on the dimensions of the denatured state and the lack of correlation with structure suggest that the radius of gyration can be a misleading reaction coordinate for unfolding/folding. Various unfolding and refolding events are observed in the denaturation simulations. In some cases, the transitions are facilitated through interactions with other portions of the protein—contact-assisted helix formation. In the native simulations, the E domain is very stable: after 6 ns, the Cα root-mean-square deviation from the starting structure is less than 1.4 Å. In contrast, the native state of the B domain deviates more and its inter-helical angles fluctuate. In apparent contrast, we note that the B domain is thermodynamically more stable than the E domain. The simulations suggest that the increased stability of the B domain may be due to heightened mobility, and therefore entropy, in the native state and decreased mobility/entropy in the more compact denatured state.

Structural characterization of an engineered tandem repeat contrasts the importance of context and sequence in protein folding

To test a different approach to understanding the relationship between the sequence of part of a protein and its conformation in the overall folded structure, the amino acid sequence corresponding to an α-helix of T4 lysozyme was duplicated in tandem. The presence of such a sequence repeat provides the protein with “choices” during folding. The mutant protein folds with almost wild-type stability, is active, and crystallizes in two different space groups, one isomorphous with wild type and the other with two molecules in the asymmetric unit. The fold of the mutant is essentially the same in all cases, showing that the inserted segment has a well-defined structure. More than half of the inserted residues are themselves helical and extend the helix present in the wild-type protein. Participation of additional duplicated residues in this helix would have required major disruption of the parent structure. The results clearly show that the residues within the duplicated sequence tend to maintain a helical conformation even though the packing interactions with the remainder of the protein are different from those of the original helix. It supports the hypothesis that the structures of individual α-helices are determined predominantly by the nature of the amino acids within the helix, rather than the structural environment provided by the rest of the protein.

Evidence that a prominent cavity in the coiled coil of HIV type 1 gp41 is an attractive drug target

Synthetic C peptides, corresponding to the C helix of the HIV type 1 (HIV-1) gp41 envelope protein, are potent inhibitors of HIV-1 membrane fusion. One such peptide is in clinical trials. The crystal structure of the gp41 core, in its proposed fusion-active conformation, is a trimer of helical hairpins in which three C helices pack against a central coiled coil. Each C helix shows especially prominent contacts with one of three symmetry-related, hydrophobic cavities on the surface of the coiled coil. We show that the inhibitory activity of the C peptide C34 depends on its ability to bind to this coiled-coil cavity. Moreover, examining a series of C34 peptide variants with modified cavity-binding residues, we find a linear relationship between the logarithm of the inhibitory potency and the stability of the corresponding helical-hairpin complexes. Our results provide strong evidence that this coiled-coil cavity is a good drug target and clarify the mechanism of C peptide inhibition. They also suggest simple, quantitative assays for the identification and evaluation of analogous inhibitors of HIV-1 entry.

Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs

SNARE [soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor] proteins are essential for membrane fusion and are conserved from yeast to humans. Sequence alignments of the most conserved regions were mapped onto the recently solved crystal structure of the heterotrimeric synaptic fusion complex. The association of the four α-helices in the synaptic fusion complex structure produces highly conserved layers of interacting amino acid side chains in the center of the four-helix bundle. Mutations in these layers reduce complex stability and cause defects in membrane traffic even in distantly related SNAREs. When syntaxin-4 is modeled into the synaptic fusion complex as a replacement of syntaxin-1A, no major steric clashes arise and the most variable amino acids localize to the outer surface of the complex. We conclude that the main structural features of the neuronal complex are highly conserved during evolution. On the basis of these features we have reclassified SNARE proteins into Q-SNAREs and R-SNAREs, and we propose that fusion-competent SNARE complexes generally consist of four-helix bundles composed of three Q-SNAREs and one R-SNARE.

Arrayed transposase-binding sequences on the ends of transposon Tn5090/Tn402

The transposon Tn5090/Tn402 encodes a 559 amino acid transposase, TniA, with a DDE motif. Gel mobility shifting and cleavage protection analysis with DNase I and hydroxyl radical probes revealed that TniA binds to multiple repeat sequences on either terminus of Tn5090/Tn402. Four of these TniA-binding 19mers occurred on the left-hand (t) end and two on the right-hand (i) end. Hydroxyl radical cleavage protection demonstrated the presence of 3–6 bp contact sequences on one face of the DNA helix. The binding pattern and organisation of repeats suggested parallels between Tn5090/Tn402 and Mu, which controls its transpositional activity in the assembly step of a higher order transpososome complex. The complex terminal structure and genes of transposase and nucleotide-binding proteins in tandem are hallmarks of the handful of Mu-like elements that are known to date.

Crystal structure of the C-terminal domain of the RAP74 subunit of human transcription factor IIF

The x-ray structure of a C-terminal fragment of the RAP74 subunit of human transcription factor (TF) IIF has been determined at 1.02-Å resolution. The α/β structure is strikingly similar to the globular domain of linker histone H5 and the DNA-binding domain of hepatocyte nuclear factor 3γ (HNF-3γ), making it a winged-helix protein. The surface electrostatic properties of this compact domain differ significantly from those of bona fide winged-helix transcription factors (HNF-3γ and RFX1) and from the winged-helix domains found within the RAP30 subunit of TFIIF and the β subunit of TFIIE. RAP74 has been shown to interact with the TFIIF-associated C-terminal domain phosphatase FCP1, and a putative phosphatase binding site has been identified within the RAP74 winged-helix domain.

The wing in yeast heat shock transcription factor (HSF) DNA-binding domain is required for full activity

The yeast heat shock transcription factor (HSF) belongs to the winged helix family of proteins. HSF binds DNA as a trimer, and additional trimers can bind DNA co-operatively. Unlike other winged helix–turn–helix proteins, HSF’s wing does not appear to contact DNA, as based on a previously solved crystal structure. Instead, the structure implies that the wing is involved in protein–protein interactions, possibly within a trimer or between adjacent trimers. To understand the function of the wing in the HSF DNA-binding domain, a Saccharomyces cerevisiae strain was created that expresses a wingless HSF protein. This strain grows normally at 30°C, but shows a decrease in reporter gene expression during constitutive and heat-shocked conditions. Removal of the wing does not affect the stability or trimeric nature of a protein fragment containing the DNA-binding and trimerization domains. Removal of the wing does result in a decrease in DNA-binding affinity. This defect was mainly observed in the ability to form the first trimer-bound complex, as the formation of larger complexes is unaffected by the deletion. Our results suggest that the wing is not involved in the highly co-operative nature of HSF binding, but may be important in stabilizing the first trimer bound to DNA.

c-Jun interacts with the corepressor TG-interacting factor (TGIF) to suppress Smad2 transcriptional activity

The Sma and Mad related (Smad) family proteins are critical mediators of the transforming growth factor-β (TGF-β) superfamily signaling. After TGF-β-mediated phosphorylation and association with Smad4, Smad2 moves to the nucleus and activates expression of specific genes through cooperative interactions with DNA-binding proteins, including members of the winged-helix family of transcription factors, forkhead activin signal transducer (FAST)-1 and FAST2. TGF-β has also been described to activate other signaling pathways, such as the c-Jun N-terminal Kinase (JNK) pathway. Here, we show that activation of JNK cascade blocked the ability of Smad2 to mediate TGF-β-dependent activation of the FAST proteins. This inhibitory activity is mediated through the transcriptional factor c-Jun, which enhances the association of Smad2 with the nuclear transcriptional corepressor TG-interacting factor (TGIF), thereby interfering with the assembly of Smad2 and the coactivator p300 in response to TGF-β signaling. Interestingly, c-Jun directly binds to the nuclear transcriptional corepressor TGIF and is required for TGIF-mediated repression of Smad2 transcriptional activity. These studies thus reveal a mechanism for suppression of Smad2 signaling pathway by JNK cascade through transcriptional repression.

Crystal structure of the SarR protein from Staphylococcus aureus

The expression of virulence determinants in Staphylococcus aureus is controlled by global regulatory loci (e.g., sarA and agr). The sar (Staphylococcus accessory regulator) locus is composed of three overlapping transcripts (sarA P1, P3, and P2, transcripts initiated from the P1, P3, and P2 promoters, respectively), all encoding the 124-aa SarA protein. The level of SarA, the major regulatory protein, is partially controlled by the differential activation of the sarA promoters. We previously partially purified a 13.6-kDa protein, designated SarR, that binds to the sarA promoter region to down-modulate sarA transcription from the P1 promoter and subsequently SarA expression. SarR shares sequence similarity to SarA, and another SarA homolog, SarS. Here we report the 2.3 Å-resolution x-ray crystal structure of the dimeric SarR-MBP (maltose binding protein) fusion protein. The structure reveals that the SarR protein not only has a classic helix–turn–helix module for DNA binding at the major grooves, but also has an additional loop region involved in DNA recognition at the minor grooves. This interaction mode could represent a new functional class of the “winged helix” family. The dimeric SarR structure could accommodate an unusually long stretch of ≈27 nucleotides with two or four bending points along the course, which could lead to the bending of DNA by 90° or more, similar to that seen in the catabolite activator protein (CAP)–DNA complex. The structure also demonstrates the molecular basis for the stable dimerization of the SarR monomers and possible motifs for interaction with other proteins.

The Three-Dimensional Structure of Aspergillus niger Pectin Lyase B at 1.7-Å Resolution1

The three-dimensional structure of Aspergillus niger pectin lyase B (PLB) has been determined by crystallographic techniques at a resolution of 1.7 Å. The model, with all 359 amino acids and 339 water molecules, refines to a final crystallographic R factor of 16.5%. The polypeptide backbone folds into a large right-handed cylinder, termed a parallel β helix. Loops of various sizes and conformations protrude from the central helix and probably confer function. The largest loop of 53 residues folds into a small domain consisting of three antiparallel β strands, one turn of an α helix, and one turn of a 310 helix. By comparison with the structure of Erwinia chrysanthemi pectate lyase C (PelC), the primary sequence alignment between the pectate and pectin lyase subfamilies has been corrected and the active site region for the pectin lyases deduced. The substrate-binding site in PLB is considerably less hydrophilic than the comparable PelC region and consists of an extensive network of highly conserved Trp and His residues. The PLB structure provides an atomic explanation for the lack of a catalytic requirement for Ca2+ in the pectin lyase family, in contrast to that found in the pectate lyase enzymes. Surprisingly, however, the PLB site analogous to the Ca2+ site in PelC is filled with a positive charge provided by a conserved Arg in the pectin lyases. The significance of the finding with regard to the enzymatic mechanism is discussed.