Comprehensive alanine-scanning mutagenesis of Escherichia coli CsrA defines two subdomains of critical functional importance

The RNA-binding protein CsrA (carbon storage regulator) of Escherichia coli is a global regulator of gene expression and is representative of the CsrA/RsmA family of bacterial proteins. These proteins act by regulating mRNA translation and stability and are antagonized by binding to small noncoding RNAs. Although the RNA target sequence and structure for CsrA binding have been well defined, little information exists concerning the protein requirements for RNA recognition. The three-dimensional structures of three CsrA/RsmA proteins were recently solved, revealing a novel protein fold consisting of two interdigitated monomers. Here, we performed comprehensive alanine-scanning mutagenesis on csrA of E. coli and tested the 58 resulting mutants for regulation of glycogen accumulation, motility, and biofilm formation. Quantitative effects of these mutations on expression of glgCA'-'lacZ, flhDC'-'lacZ, and pgaA'-'lacZ translational fusions were also examined, and eight of the mutant proteins were purified and tested for RNA binding. These studies identified two regions of the amino acid sequence that were critical for regulation and RNA binding, located within the first (beta1, residues 2-7) and containing the last (beta5, residues 40-47) beta-strands of CsrA. The beta1 and beta5 strands of opposite monomers lie adjacent and parallel to each other in the three-dimensional structure of this protein. Given the symmetry of the CsrA dimer, these findings imply that two distinct RNA binding surfaces or functional subdomains lie on opposite sides of the protein.     

Folding and unfolding of gammaTIM monomers and dimers

Kinetic simulations of the folding and unfolding of triosephosphate isomerase (TIM) from yeast were conducted using a single monomer gammaTIM polypeptide chain that folds as a monomer and two gammaTIM chains that fold to the native dimer structure. The basic protein model used was a minimalist Gō model using the native structure to determine attractive energies in the protein chain. For each simulation type--monomer unfolding, monomer refolding, dimer unfolding, and dimer refolding--thirty simulations were conducted, successfully capturing each reaction in full. Analysis of the simulations demonstrates four main conclusions. First, all four simulation types have a similar "folding order", i.e., they have similar structures in intermediate stages of folding between the unfolded and folded state. Second, despite this similarity, different intermediate stages are more or less populated in the four different simulations, with 1), no intermediates populated in monomer unfolding; 2), two intermediates populated with beta(2)-beta(4) and beta(1)-beta(5) regions folded in monomer refolding; 3), two intermediates populated with beta(2)-beta(3) and beta(2)-beta(4) regions folded in dimer unfolding; and 4), two intermediates populated with beta(1)-beta(5) and beta(1)-beta(5) + beta(6) + beta(7) + beta(8) regions folded in dimer refolding. Third, simulations demonstrate that dimer binding and unbinding can occur early in the folding process before complete monomer-chain folding. Fourth, excellent agreement is found between the simulations and MPAX (misincorporation proton alkyl exchange) experiments. In total, this agreement demonstrates that the computational Gō model is accurate for gammaTIM and that the energy landscape of gammaTIM appears funneled to the native state.     

Quaternary interactions in eye lens beta-crystallins: basic and acidic subunits of beta-crystallins favor heterologous association

beta-Crystallins are complex eye lens proteins made up of several related basic and acidic subunits that combine to form differently sized oligomers each displaying extensive polydispersity. As the sequences are homologous to the X-ray-determined bilobal structure of gamma-crystallin, beta-subunits are visualized as having a similar structure with additional N- and C-terminal extensions. Two basic (beta B2 and beta B3) and two acidic (beta A3 and beta A4) subunits have been isolated in deaggregating media, refolded, and reassociated in various combinations to determine which components favor dimers or higher oligomers. Homopolymers were compared with beta B2 homodimer in terms of charge, using Mono Q fast protein liquid chromatography, and size, using Superose 12 chromatography. Heterooligomeric formations were monitored by their intermediate charge properties compared with homooligomers. beta B2 associates with either beta B3- or beta A4-forming heterodimers whereas a larger oligomer is formed with beta A3. Naturally occurring beta-crystallin oligomers were analyzed by Mono Q chromatography and PhastGel electrophoresis. Whereas beta B2, beta B3, and beta A4 can each be reassociated to homodimers, beta A4 dimers are not found in native beta-crystallins. beta B2-beta A3 is a major component of intermediate-sized beta L1-crystallin and is absent from dimeric beta L2-crystallin. It is suggested that the pH dependence of the size of beta L1-crystallin is due to a dimer to tetramer equilibrium. By following dimer interactions using Superose 12 chromatography, beta B2-beta A4 was shown to interact with beta B2-beta A3. A model of beta-crystallin structure is proposed based on beta-subunits forming dimers with the next level of organization requiring an acidic subunit, beta A3, with a long N-terminal extension.     

Complex assembly mechanism and an RNA-binding mode of the human p14-SF3b155 spliceosomal protein complex identified by NMR solution structure and functional analyses

The spliceosomal protein p14, a component of the SF3b complex in the U2 small nuclear ribonucleoprotein (snRNP), is essential for the U2 snRNP to recognize the branch site adenosine. The elucidation of the dynamic process of the splicing machinery rearrangement awaited the solution structural information. We identified a suitable complex of human p14 and the SF3b155 fragment for the determination of its solution structure by NMR. In addition to the overall structure of the complex, which was recently reported in a crystallographic study (typical RNA recognition motif fold beta1-alpha1-beta2-beta3-alpha2-beta4 of p14, and alphaA-betaA fold of the SF3b155 fragment), we identified three important features revealed by the NMR solution structure. First, the C-terminal extension and the nuclear localization signal of p14 (alpha3 and alpha4 in the crystal structure, respectively) were dispensable for the complex formation. Second, the proline-rich segment of SF3b155, following betaA, closely approaches p14. Third, interestingly, the beta1-alpha1 loop and the alpha2-beta4 beta-hairpin form a positively charged groove. Extensive mutagenesis analyses revealed the functional relevance of the residues involved in the protein-protein interactions: two aromatic residues of SF3b155 (Phe408 and Tyr412) play crucial roles in the complex formation, and two hydrophobic residues (Val414 and Leu415) in SF3b 155 serve as an anchor for the complex formation, by cooperating with the aromatic residues. These findings clearly led to the conclusion that SFb155 binds to p14 with three contact points, involving Phe408, Tyr412, and Val414/Leu415. Furthermore, to dissect the interactions between p14 and the branch site RNA, we performed chemical-shift-perturbation experiments, not only for the main-chain but also for the side-chain resonances, for several p14-SF3b155 complex constructs upon binding to RNA. These analyses identified a positively charged groove and the C-terminal extension of p14 as RNA-binding sites. Strikingly, an aromatic residue in the beta1-alpha1 loop, Tyr28, and a positively charged residue in the alpha2-beta4 beta-hairpin, Agr85, are critical for the RNA-binding activity of the positively charged groove. The Tyr28Ala and Arg85Ala point mutants and a deletion mutant of the C-terminal extension clearly revealed that their RNA binding activities were independent of each other. Collectively, this study provides details for the protein-recognition mode of p14 and insight into the branch site recognition.     

Solution structures of the first and second RNA-binding domains of human U2 small nuclear ribonucleoprotein particle auxiliary factor (U2AF(65)).

The large subunit of the human U2 small nuclear ribonucleoprotein particle auxiliary factor (hU2AF(65)) is an essential RNA-splicing factor required for the recognition of the polypyrimidine tract immediately upstream of the 3' splice site. In the present study, we determined the solution structures of two hU2AF(65) fragments, corresponding to the first and second RNA-binding domains (RBD1 and RBD2, respectively), by nuclear magnetic resonance spectroscopy. The tertiary structure of RBD2 is similar to that of typical RNA-binding domains with the beta1-alpha1-beta2-beta3-alpha2-beta4 topology. In contrast, the hU2AF(65) RBD1 structure has unique features: (i) the alpha1 helix is elongated by one turn toward the C-terminus; (ii) the loop between alpha1 and beta2 (the alpha1/beta2 loop) is much longer and has a defined conformation; (iii) the beta2 strand is (188)AVQIN(192), which was not predicted by sequence alignments; and (iv) the beta2/beta3 loop is much shorter. Chemical shift perturbation experiments showed that the U2AF-binding RNA fragments interact with the four beta-strands of RBD2 whereas, in contrast, they interact with beta1, beta3 and beta4, but not with beta2 or the alpha1/beta2 loop, of RBD1. The characteristic alpha1-beta2 structure of the hU2AF(65) RBD1 may interact with other proteins, such as UAP56.     

A NMR guided approach for CsrA–RNA crystallization

Structure determination of protein–nucleic acid complexes remains a challenging task. Here we present a simple method for generating crystals of a CsrA–nucleic acid complex, guided entirely by results from nuclear magnetic resonances spectroscopy (NMR) spectroscopy. Using a construct that lacks thirteen non-essential C-terminal residues, efficient binding to DNA could be demonstrated. One CsrA dimer interacts with two DNA oligonucleotides, similar to previous findings with RNA. Furthermore, the NMR study of the CsrA–DNA complex was the basis for successfully homing in on conditions that were suitable for obtaining crystals of the CsrA–DNA complex. Our results may be useful for those cases where RNA in protein–nucleic acid complexes may be replaced by DNA.     

Olfactory marker protein (OMP) exhibits a beta-clam fold in solution: implications for target peptide interaction and olfactory signal transduction

Olfactory marker protein (OMP) is a ubiquitous, cytoplasmic protein found in mature olfactory receptor neurons of all vertebrates. Electrophysiological and behavioral studies demonstrate that it is a modulator of the olfactory signal transduction pathway. Here, we demonstrate that the solution structure of OMP, as determined by NMR studies, is a single globular domain protein comprised of eight beta-strands forming two beta-sheets oriented orthogonally to one another, thus exhibiting a "beta-clam" or "beta-sandwich" fold: beta-sheet 1 is comprised of beta3-beta8-beta1-beta2 and beta-sheet 2 contains beta6-beta5-beta4-beta7. Insertions include two, long alpha-helices located on opposite sides of the beta-clam and three flexible loops. The juxtaposition of beta-strands beta6-beta5-beta4-beta7-beta2-beta1-beta8-beta3 forms a continuously curved surface and encloses one side of the beta-clam. The "cleft" formed by the two beta-sheets is opposite to the closed end of the beta-clam. Using a peptide titration series, we have identified this cleft as the binding surface for a peptide derived from the Bex1 protein. The highly conserved Omega-loop structure adjacent to the Bex1 peptide-binding surface found in OMP may be the site of additional OMP-protein interactions related to its role in modulating olfactory signal transduction. Thus, the interaction between the OMP and Bex1 proteins could facilitate the interaction between OMP and other components of the olfactory signaling pathway.     

Structure and dynamics of an acid-denatured protein G mutant

NMR studies of protein denatured states provide insights into potential initiation sites for folding that may be too transient to be observed kinetically. We have characterized the structure and dynamics of the acid-denatured state of protein G by using a F30H mutant of G(B1) which is on the margin of stability. At 5 degrees C, F30H-G(B1) is greater than 95% folded at pH 7.0 and is greater than 95% unfolded at pH 4.0. This range of stability is useful because the denatured state can be examined under relatively mild conditions which are optimal for folding G(B1). We have assigned almost all backbone (15)N, H(N), and H(alpha) resonances in the acid-denatured state. Chemical shift, coupling constant, and NOE data indicate that the denatured state has considerably more residual structure when studied under these mild conditions than in the presence of chemical denaturants. The acid-denatured state populates nativelike conformations with both alpha-helical and beta-hairpin characteristics. To our knowledge, this is the first example of a denatured state with NOE and coupling constant evidence for beta-hairpin character. A number of non-native turn structures are also detected, particularly in the region corresponding to the beta1-beta2 hairpin of the folded state. Steady-state ?(1)H-(15)N? NOE results demonstrate restricted backbone flexibility in more structured regions of the denatured protein. Overall, our studies suggest that regions of the helix, the beta3-beta4 hairpin, and the beta1-beta2 turn may serve as potential initiation sites for folding of G(B). Furthermore, residual structure in acid-denatured F30H-G(B1) is more extensive than in peptide fragments corresponding to the beta1-beta2, alpha-helix, and beta3-beta4 regions, suggesting additional medium-to-long-range interactions in the full-length polypeptide chain.     

Solution NMR structure of ribosome-binding factor A (RbfA), a cold-shock adaptation protein from Escherichia coli

Ribosome-binding factor A (RbfA) from Escherichia coli is a cold-shock adaptation protein. It is essential for efficient processing of 16S rRNA and is suspected to interact with the 5'-terminal helix (helix I) of 16S rRNA. RbfA is a member of a large family of small proteins found in most bacterial organisms, making it an important target for structural proteomics. Here, we describe the three-dimensional structure of RbfADelta25, a 108 residue construct with 25 residues removed from the carboxyl terminus of full-length RbfA, determined in solution at pH 5.0 by heteronuclear NMR methods. The structure determination was carried out using largely automated methods for determining resonance assignments, interpreting nuclear Overhauser effect (NOE) spectroscopy (NOESY) spectra, and structure generation. RbfADelta25 has an alpha+beta fold containing three helices and three beta-strands, alpha1-beta1-beta2-alpha2-alpha3-beta3. The structure has type-II KH-domain fold topology, related to conserved KH sequence family proteins whose betaalphaalphabeta subunits are characterized by a helix-turn-helix motif with sequence signature GxxG at the turn. In RbfA, this betaalphaalphabeta subunit is characterized by a helix-kink-helix motif in which the GxxG sequence is replaced by a conserved AxG sequence, including a strongly conserved Ala residue at position 75 forming an interhelical kink. The electrostatic field distribution about RbfADelta25 is bipolar; one side of the molecule is strongly negative and the opposite face has a strong positive electrostatic field. A "dynamic hot spot" of RbfADelta25 has been identified in the vicinity of a beta-bulge at strongly conserved residue Ser39 by 15N R(1), R(2) relaxation rate and heteronuclear 15N-1H NOE measurements. Analyses of these distributions of electrostatic field and internal dynamics, together with evolutionary implications of fold and sequence conservation, suggest that RbfA is indeed a nucleic acid-binding protein, and identify a potential RNA-binding site in or around the conserved polypeptide segment Ser76-Asp100 corresponding to the alpha3-loop-beta3 helix-loop-strand structure. While the structure of RbfADelta25 is most similar to that of the KH domain of the E.coli Era GTPase, its electrostatic field distribution is most similar to the KH1 domain of the NusA protein from Thermotoga maritima, another cold-shock associated RNA-binding protein. Both RbfA and NusA are regulated in the same E.coli operon. Structural and functional similarities between RbfA, NusA, and other bacterial type II KH domains suggest previously unsuspected evolutionary relationships between these cold-shock associated proteins.     

Apparent local stability of the secondary structure of Azotobacter vinelandii holoflavodoxin II as probed by hydrogen exchange: implications for redox potential regulation and flavodoxin folding.

As a first step to determine the folding pathway of a protein with an alpha/beta doubly wound topology, the 1H, 13C, and 15N backbone chemical shifts of Azotobacter vinelandii holoflavodoxin II (179 residues) have been determined using multidimensional NMR spectroscopy. Its secondary structure is shown to contain a five-stranded parallel beta-sheet (beta2-beta1-beta3-beta4-beta5) and five alpha-helices. Exchange rates for the individual amide protons of holoflavodoxin were determined using the hydrogen exchange method. The amide protons of 65 residues distributed throughout the structure of holoflavodoxin exchange slowly at pH* 6.2 [kex < 10(-5) s(-1)] and can be used as probes in future folding studies. Measured exchange rates relate to apparent local free energies for transient opening. We propose that the amide protons in the core of holoflavodoxin only exchange by global unfolding of the apo state of the protein. The results obtained are discussed with respect to their implications for flavodoxin folding and for modulation of the flavin redox potential by the apoprotein. We do not find any evidence that A. vinelandii holoflavodoxin II is divided into two subdomains based on its amide proton exchange rates, as opposed to what is found for the structurally but not sequentially homologous alpha/beta doubly wound protein Che Y.     

Crystal structures of Nova-1 and Nova-2 K-homology RNA-binding domains.

BACKGROUND: Nova-1 and Nova-2 are related neuronal proteins that were initially cloned using antisera obtained from patients with the autoimmune neurological disease paraneoplastic opsoclonus-myoclonus ataxia (POMA). Both of these disease gene products contain three RNA-binding motifs known as K-homology or KH domains, and their RNA ligands have been identified via binding-site selection experiments. The KH motif structure has been determined previously using NMR spectroscopy, but not using X-ray crystallography. Many proteins contain more than one KH domain, yet there is no published structural information regarding the behavior of such multimers. RESULTS: We have obtained the first X-ray crystallographic structures of KH-domain-containing proteins. Structures of the third KH domains (KH3) of Nova-1 and Nova-2 were determined by multiple isomorphous replacement and molecular replacement at 2.6 A and 2.0 A, respectively. These highly similar RNA-binding motifs form a compact protease-resistant domain resembling an open-faced sandwich, consisting of a three-stranded antiparallel beta sheet topped by three alpha helices. In both Nova crystals, the lattice is composed of symmetric tetramers of KH3 domains that are created by two dimer interfaces. CONCLUSIONS: The crystal structures of both Nova KH3 domains are similar to the previously determined NMR structures. The most significant differences among the KH domains involve changes in the positioning of one or more of the alpha helices with respect to the betasheet, particularly in the NMR structure of the KH1 domain of the Fragile X disease protein FMR-1. Loop regions in the KH domains are clearly visible in the crystal structure, unlike the NMR structures, revealing the conformation of the invariant Gly-X-X-Gly segment that is thought to participate in RNA-binding and of the variable region. The tetrameric arrangements of the Nova KH3 domains provide insights into how KH domains may interact with each other in proteins containing multiple KH motifs.     

A test of the relationship between sequence and structure in proteins: excision of the heme binding site in apocytochrome b5.

The water-soluble domain of rat hepatic holocytochrome b5 is an alphabeta protein containing elements of secondary structure in the sequence beta1-alpha1-beta4-beta3-alpha2-alpha3-beta5- alpha4-alpha5-beta2-alpha6. The heme group is enclosed by four helices, a2, a3, a4, and a5. To test the hypothesis that a small b hemoprotein can be constructed in two parts, one forming the heme site, the other an organizing scaffold, a protein fragment corresponding to beta1-alpha1-beta4-beta3-lambda-beta2-alpha6 was prepared, where lambda is a seven-residue linker bypassing the heme binding site. The fragment ("abridged b5") was found to contain alpha and beta secondary structure by circular dichroism spectroscopy and tertiary structure by Trp fluorescence emission spectroscopy. NMR data revealed a species with spectral properties similar to those of the full-length apoprotein. This folded form is in slow equilibrium on the chemical shift time scale with other less folded species. Thermal denaturation, as monitored by circular dichroism, absorption, and fluorescence spectroscopy, as well as size-exclusion chromatography-fast protein liquid chromatography (SEC-FPLC), confirmed the coexistence of at least two distinct conformational ensembles. It was concluded that the protein fragment is capable of adopting a specific fold likely related to that of cytochrome b5, but does not achieve high thermodynamic stability and cooperativity. Abridged b5 demonstrates that the spliced sequence contains the information necessary to fold the protein. It suggests that the dominating influence to restrict the conformational space searched by the chain is structural propensities at a local level rather than internal packing. The sequence also holds the properties necessary to generate a barrier to unfolding.     

Folding mechanisms of proteins with high sequence identity but different folds

The problem of how a protein folds from a linear chain of amino acids to the three-dimensional structure necessary for function is often investigated using proteins with a low degree of sequence identity that adopt different folds. The design of pairs of proteins with a high degree of sequence identity but different folds offers the opportunity for a complementary study; in two highly similar sequences, which residues are the most important in directing folding to a particular structure? Here we use molecular dynamics simulations to characterize the folding-unfolding pathways of a pair of proteins designed by Bryan and co-workers [Alexander, P. A., et al. (2005) Biochemistry 44, 14045-14054; He, Y. N., et al. (2005) Biochemistry 44, 14055-14061]. Despite being 59% identical, the two protein sequences fold to two different structures. The first sequence folds to the alpha+beta protein G structure and the second to the all-alpha-helical protein A structure. We show that the final protein structure is determined early along the folding pathway. In folding to the protein G structure, the single alpha-helix (alpha1) and the beta3-beta4 turn fold early. Formation of the hairpin turn essentially prevents folding to helical structure in this region of the protein. This early structure is then consolidated by formation of long-range hydrophobic interactions between alpha1 and the beta3-beta4 turn. The protein A sequence differs both in the residues that form the beta3-beta4 turn and also in many of the residues that form the early hydrophobic interactions in the protein G structure. Instead, in the protein A sequence, a more hierarchical mechanism is observed, with helices folding before many of the tertiary interactions are formed. We find that small, but critical, sequence differences determine the topology of the protein early along the folding pathway, which help to explain the process by which one fold can evolve into another.     

Solution structure, stability, and nucleic acid binding of the hyperthermophile protein Sso10b2

The Sso10b (or Alba) family of proteins is a conserved group of archaeal and eukaryotic proteins which are thought to play a role in both chromatin organization and RNA metabolism. We describe here the solution structure and properties of Sso10b2 from Sulfolobus solfataricus. NMR data including residual dipolar couplings and (15)N relaxation data demonstrated that the protein adopts a beta(1)alpha(1)beta(2)alpha(2)beta(3)beta(4) topology with an IF-3-like fold. The protein dimerizes in solution at 30 degrees C via a hydrophobic surface defined by the C-terminal alpha(2)beta(3)beta(4) elements with a structure similar to one of the putative dimers indicated by previous crystal structures. DSC and circular dichroism data demonstrated an unusual two-state structural transition near the growth temperature which led to an increase in beta-sheet content without dissociation of the dimer. The cooperativity of the transition exceeded that of a dimer at pH 7, demonstrating the presence of higher order oligomers near the growth temperature at pH 7. Reverse titrations of Sso10b2 with nucleic acid showed that the protein binds single-stranded DNA (K(d) of 3 x 10(-)(7) M) with higher affinity than RNA (1.3 x 10(-)(6) M) or double-stranded DNA (1.5 x 10(-)(5) M) in 10 mM KH(2)PO(4) (pH 7.0, 20 degrees C). NMR chemical shift perturbation data indicated that single-stranded DNA and RNA binding occurred across the same dimer interface and encompassed a surface defined by the C-terminal ends of the beta(1), beta(2), and beta(3) strands of each monomer.     

Solution structure of the hypothetical protein TA0095 from Thermoplasma acidophilum: a novel superfamily with a two-layer sandwich architecture

TA0095 is a 96-residue hypothetical protein from Thermoplasma acidophilum that exhibits no sequence similarity to any protein of known structure. Also, TA0095 is a member of the COG4004 orthologous group of unknown function found in Archaea bacteria. We determined its three-dimensional structure by NMR methods. The structure displays an alpha/beta two-layer sandwich architecture formed by three alpha-helices and five beta-strands following the order beta1-alpha1-beta2-beta3-beta4-beta5-alpha2-alpha3. Searches for structural homologs indicate that the TA0095 structure belongs to the TBP-like fold, constituting a novel superfamily characterized by an additional C-terminal helix. The TA0095 structure provides a fold common to the COG4004 proteins that will obviously belong to this new superfamily. Most hydrophobic residues conserved in the COG4004 proteins are buried in the structure determined herein, thus underlying their importance for structure stability. Considering that the TA0095 surface shows a large positively charged patch with a high degree of residue conservation within the COG4004 domain, the biological function of TA0095 and the rest of COG4004 proteins might occur through binding a negatively charged molecule. Like other TBP-like fold proteins, the COG4004 proteins might be DNA-binding proteins. The fact that TA0095 is shown to interact with large DNA fragments is in favor of this hypothesis, although nonspecific DNA binding cannot be ruled out.     

Solution structure and dynamics of a designed monomeric variant of the lambda Cro repressor.

The solution structure of a monomeric variant of the lambda Cro repressor has been determined by multidimensional NMR. Cro K56[DGEVK] differs from wild-type Cro by the insertion of five amino acids at the center of the dimer interface. 1H and 15N resonances for 70 of the 71 residues have been assigned. Thirty-two structures were calculated by hybrid distance geometry/simulated annealing methods using 463 NOE-distance restraints, 26 hydrogen-bond, and 39 dihedral-angle restraints. The root-mean-square deviation (RMSD) from the average structure for atoms in residues 3-60 is 1.03 +/- 0.44 A for the peptide backbone and 1.6 +/- 0.73 A for all nonhydrogen atoms. The overall structure conforms very well to the original design. Although the five inserted residues form a beta hairpin as expected, this engineered turn as well as other turns in the structure are not well defined by the NMR data. Dynamics studies of backbone amides reveal T1/T2 ratios of residues in the alpha2-alpha3, beta2-beta3, and engineered turn that are reflective of chemical exchange or internal motion. The solution structure and dynamics are discussed in light of the conformational variation that has been observed in other Cro structures, and the importance of flexibility in DNA recognition.     

The 1.1 A crystal structure of human TGF-beta type II receptor ligand binding domain

Transforming growth factor beta (TGF-beta) is involved in a wide range of biological functions including development, carcinogenesis, and immune regulation. Here we report the 1.1 A resolution crystal structure of human TGF-beta type II receptor ectodomain (TBRII). The overall structure of TBRII is similar to that of activin type II receptor ectodomain (ActRII) and bone morphogenic protein receptor type IA (BRIA). It displays a three-finger toxin fold with fingers formed by the beta strand pairs beta1-beta2, beta3-beta4, and beta5-beta6. The first finger in the TBRII is significantly longer than in ActRII and BRIA and folds tightly between the second finger and the C terminus. Surface charge distributions and hydrophobic patches predict potential TBRII binding sites.