Lisa R. Warner

Structure of the BamC two-domain protein obtained by Rosetta with a limited NMR data set

The CS-RDC-NOE Rosetta program was used to generate the solution structure of a 27-kDa fragment of the Escherichia coli BamC protein from a limited set of NMR data. The BamC protein is a component of the essential five-protein β-barrel assembly machine in E. coli. The first 100 residues in BamC were disordered in solution. The Rosetta calculations showed that BamC₁₀₁₋₃₄₄ forms two well-defined domains connected by an ~18-residue linker, where the relative orientation of the domains was not defined. Both domains adopt a helix-grip fold previously observed in the Bet v 1 superfamily. ¹⁵N relaxation data indicated a high degree of conformational flexibility for the linker connecting the N-terminal domain and the C-terminal domain in BamC. The results here show that CS-RDC-NOE Rosetta is robust and has a high tolerance for misassigned nuclear Overhauser effect restraints, greatly simplifying NMR structure determinations.

Transient electrostatic interactions dominate the conformational equilibrium sampled by multidomain splicing factor U2AF65: a combined NMR and SAXS study.

Multidomain proteins containing intrinsically disordered linkers exhibit large-scale dynamic modes that play key roles in a multitude of molecular recognition and signaling processes. Here, we determine the conformational space sampled by the multidomain splicing factor U2AF65 using complementary nuclear magnetic resonance spectroscopy and small-angle scattering data. Available degrees of conformational freedom are initially stochastically sampled and experimental data then used to delineate the potential energy landscape in terms of statistical probability. The spatial distribution of U2AF65 conformations is found to be highly anisotropic, comprising significantly populated interdomain contacts that appear to be electrostatic in origin. This hypothesis is supported by the reduction of signature PREs reporting on expected interfaces with increasing salt concentration. The described spatial distribution reveals the complete spectrum of the unbound forms of U2AF65 that coexist with the small percentage of a preformed RNA-bound domain arrangement required for polypyrimidine-tract recognition by conformational selection. More generally, the proposed approach to describing conformational equilibria of multidomain proteins can be further combined with other experimental data that are sensitive to domain dynamics.

Phosphorylation releases constraints to domain motion in ERK2

Significance This paper uses NMR methods to compare the dynamics of a protein kinase in its active and inactive states. The results show that domain movements in the MAP kinase ERK2 are inherently constrained until the enzyme is activated by phosphorylation, with the constraint located at the hinge region. This represents an important mode for dynamical regulation in ERK2, not anticipated from previous X-ray structural analyses. Protein motions control enzyme catalysis through mechanisms that are incompletely understood. Here NMR 13C relaxation dispersion experiments were used to monitor changes in side-chain motions that occur in response to activation by phosphorylation of the MAP kinase ERK2. NMR data for the methyl side chains on Ile, Leu, and Val residues showed changes in conformational exchange dynamics in the microsecond-to-millisecond time regime between the different activity states of ERK2. In inactive, unphosphorylated ERK2, localized conformational exchange was observed among methyl side chains, with little evidence for coupling between residues. Upon dual phosphorylation by MAP kinase kinase 1, the dynamics of assigned methyls in ERK2 were altered throughout the conserved kinase core, including many residues in the catalytic pocket. The majority of residues in active ERK2 fit to a single conformational exchange process, with kex ≈ 300 s−1 (kAB ≈ 240 s−1/kBA ≈ 60 s−1) and pA/pB ≈ 20%/80%, suggesting global domain motions involving interconversion between two states. A mutant of ERK2, engineered to enhance conformational mobility at the hinge region linking the N- and C-terminal domains, also induced two-state conformational exchange throughout the kinase core, with exchange properties of kex ≈ 500 s−1 (kAB ≈ 15 s−1/kBA ≈ 485 s−1) and pA/pB ≈ 97%/3%. Thus, phosphorylation and activation of ERK2 lead to a dramatic shift in conformational exchange dynamics, likely through release of constraints at the hinge.

Structural model of the amino propeptide of collagen XI α1 chain with similarity to the LNS domains

Fibrillar collagens are the principal structural molecules of connective tissues. The assembly of collagen fibrils is regulated by quantitatively minor fibrillar collagens, types V and XI. A unique amino‐terminal propeptide domain of these collagens has been attributed this regulatory role. The structure of the amino terminal propeptide has yet to be determined. Low sequence similarity necessitated a secondary structure‐based method to carry out homology modeling based upon the determined structure of LNS family members, named for a common structure in the laminin LG5 domain, the neurexin 1B domain and the sex hormone binding globulin. Distribution of amino acids within the model suggested glycosaminoglycan interaction and calcium binding. These activities were tested experimentally. Sequence analyses of existing genes for collagens indicate that 16 known collagen α chains may contain an LNS domain. A similar approach may prove useful for structure/function studies of similar domains in other collagens with similar domains. This will provide mechanistic details of the organization and assembly of the extracellular matrix and the underlying basis of structural integrity in connective tissues. The absolute requirement for collagen XI in skeletal growth is indicated by collagen XI deficiencies such as chondrodystrophies found in the cho/cho mouse and in humans with Stickler syndrome.

Isoform-specific Heparan Sulfate Binding within the Amino-terminal Noncollagenous Domain of Collagen α1(XI)

Collagen type XI is a constituent of the pericellular matrix of chondrocytes and plays a role in the regulation of fibrillogenesis. The amino-terminal domain of collagen type XI α1 chain is a noncollagenous structure that has been identified on the surface of cartilage collagen fibrils. The biochemical composition of the amino-terminal domain varies due to alternative splicing of the primary transcript. Recombinantly expressed α1(XI) aminoterminal domain isoforms were used in this study to investigate potential interactions. Purified products were analyzed for heparan sulfate binding properties. The results demonstrated that two additional binding sites exist within the α1(XI) aminoterminal domain, one within the amino propeptide and one within the variable region of the amino-terminal domain. Analysis of relative affinities indicated that the site located within the amino propeptide (site 1) was of similar affinity to sites that exist within the major triple helix of collagen type XI. Substitution of amino acid residues 147 to 152 within the amino propeptide by site-directed mutagenesis resulted in altered affinity for heparan sulfate. The binding site located within the variable region (site 2) demonstrated significantly higher affinity than other sites within the molecule. Displacement of collagen type XI within the pericellular matrix was observed in cell culture in the presence of excess heparan sulfate and by treatment with heparinase. These studies suggest two additional binding sites located within the noncollagenous amino-terminal domain that may play a role in the function of collagen type XI. The localization of collagen type XI within the pericellular matrix may be dependent upon interactions with heparan sulfate proteoglycans, and these are likely to take place in an isoform-specfic manner.

CO2-fixing one-carbon metabolism in a cellulose-degrading bacterium Clostridium thermocellum

Significance High carbon yield in the bioengineering of heterotrophic bacteria is hindered by carbon loss to CO2 production. We provide evidence showing Clostridium thermocellum, a cellulose-degrading bacterium and a model consolidated bioprocessing organism, can fix CO2 while growing predominantly on cellobiose, a cellulose-derived disaccharide. By adding 13C-bicarbonate to the bacterial culture and tracking 13C-labeled metabolites, we discovered active reductive one-carbon (C1) metabolism in this bacterium. We further identified critical enzymes responsible for fixing CO2 and channeling the fixed carbon to the C1 metabolic pathway. Our findings pave the way to future engineering of this bacterium to use cellulose and CO2 simultaneously as a means to improve microbial carbon efficiency that is constrained by theoretical limitation and to reduce CO2 in the environment. Clostridium thermocellum can ferment cellulosic biomass to formate and other end products, including CO2. This organism lacks formate dehydrogenase (Fdh), which catalyzes the reduction of CO2 to formate. However, feeding the bacterium 13C-bicarbonate and cellobiose followed by NMR analysis showed the production of 13C-formate in C. thermocellum culture, indicating the presence of an uncharacterized pathway capable of converting CO2 to formate. Combining genomic and experimental data, we demonstrated that the conversion of CO2 to formate serves as a CO2 entry point into the reductive one-carbon (C1) metabolism, and internalizes CO2 via two biochemical reactions: the reversed pyruvate:ferredoxin oxidoreductase (rPFOR), which incorporates CO2 using acetyl-CoA as a substrate and generates pyruvate, and pyruvate-formate lyase (PFL) converting pyruvate to formate and acetyl-CoA. We analyzed the labeling patterns of proteinogenic amino acids in individual deletions of all five putative PFOR mutants and in a PFL deletion mutant. We identified two enzymes acting as rPFOR, confirmed the dual activities of rPFOR and PFL crucial for CO2 uptake, and provided physical evidence of a distinct in vivo “rPFOR-PFL shunt” to reduce CO2 to formate while circumventing the lack of Fdh. Such a pathway precedes CO2 fixation via the reductive C1 metabolic pathway in C. thermocellum. These findings demonstrated the metabolic versatility of C. thermocellum, which is thought of as primarily a cellulosic heterotroph but is shown here to be endowed with the ability to fix CO2 as well.

Recognition of the 3′ splice site RNA by the U2AF heterodimer involves a dynamic population shift

Significance The splicing of human pre-mRNAs is tightly controlled and regulated during the assembly of the spliceosome onto pre-mRNA introns. Recognition of regulatory RNA sequence motifs by splicing factors is an essential early step during spliceosome assembly. We combine single-pair FRET and NMR to show that the recognition of the 3′ splice site in pre-mRNA introns by the essential heterodimeric splicing factor U2 auxiliary factor (U2AF) involves conformational dynamics and population shifts of its RNA binding domains between open and closed conformations. Unexpectedly, the small subunit U2AF35 facilitates the recognition of weak splice sites by a population shift of the RNA binding domains of U2AF65 toward the open conformation. Notably, disease-linked mutations in U2AF65 do not affect RNA or U2AF35 binding. An essential early step in the assembly of human spliceosomes onto pre-mRNA involves the recognition of regulatory RNA cis elements in the 3′ splice site by the U2 auxiliary factor (U2AF). The large (U2AF65) and small (U2AF35) subunits of the U2AF heterodimer contact the polypyrimidine tract (Py-tract) and the AG-dinucleotide, respectively. The tandem RNA recognition motif domains (RRM1,2) of U2AF65 adopt closed/inactive and open/active conformations in the free form and when bound to bona fide Py-tract RNA ligands. To investigate the molecular mechanism and dynamics of 3′ splice site recognition by U2AF65 and the role of U2AF35 in the U2AF heterodimer, we have combined single-pair FRET and NMR experiments. In the absence of RNA, the RRM1,2 domain arrangement is highly dynamic on a submillisecond time scale, switching between closed and open conformations. The addition of Py-tract RNA ligands with increasing binding affinity (strength) gradually shifts the equilibrium toward an open conformation. Notably, the protein–RNA complex is rigid in the presence of a strong Py-tract but exhibits internal motion with weak Py-tracts. Surprisingly, the presence of U2AF35, whose UHM domain interacts with U2AF65 RRM1, increases the population of the open arrangement of U2AF65 RRM1,2 in the absence and presence of a weak Py-tract. These data indicate that the U2AF heterodimer promotes spliceosome assembly by a dynamic population shift toward the open conformation of U2AF65 to facilitate the recognition of weak Py-tracts at the 3′ splice site. The structure and RNA binding of the heterodimer was unaffected by cancer-linked myelodysplastic syndrome mutants.

Next-Generation Heteronuclear Decoupling for High-Field Biomolecular NMR Spectroscopy†

Ultra-high-field NMR spectroscopy requires an increased bandwidth for heteronuclear decoupling, especially in biomolecular NMR applications. Composite pulse decoupling cannot provide sufficient bandwidth at practical power levels, and adiabatic pulse decoupling with sufficient bandwidth is compromised by sideband artifacts. A novel low-power, broadband heteronuclear decoupling pulse is presented that generates minimal, ultra-low sidebands. The pulse was derived using optimal control theory and represents a new generation of decoupling pulses free from the constraints of periodic and cyclic sequences. In comparison to currently available state-of-the-art methods this novel pulse provides greatly improved decoupling performance that satisfies the demands of high-field biomolecular NMR spectroscopy.

Structural Analysis of Protein-RNA Complexes in Solution Using NMR Paramagnetic Relaxation Enhancements.

Biological activity in the cell is predominantly mediated by large multiprotein and protein-nucleic acid complexes that act together to ensure functional fidelity. Nuclear magnetic resonance (NMR) spectroscopy is the only method that can provide information for high-resolution three-dimensional structures and the conformational dynamics of these complexes in solution. Mapping of binding interfaces and molecular interactions along with the characterization of conformational dynamics is possible for very large protein complexes. In contrast, de novo structure determination by NMR becomes very time consuming and difficult for protein complexes larger than 30 kDa as data are noisy and sparse. Fortunately, high-resolution structures are often available for individual domains or subunits of a protein complex and thus sparse data can be used to define their arrangement and dynamics within the assembled complex. In these cases, NMR can therefore be efficiently combined with complementary solution techniques, such as small-angle X-ray or neutron scattering, to provide a comprehensive description of the structure and dynamics of protein complexes in solution. Particularly useful are NMR-derived paramagnetic relaxation enhancements (PREs), which provide long-range distance restraints (ca. 20Å) for structural analysis of large complexes and also report on conformational dynamics in solution. Here, we describe the use of PREs from sample production to structure calculation, focusing on protein-RNA complexes. On the basis of recent examples from our own research, we demonstrate the utility, present protocols, and discuss potential pitfalls when using PREs for studying the structure and dynamic features of protein-RNA complexes.

Structural basis for the recognition of spliceosomal SmN/B/B’ proteins by the RBM5 OCRE domain in splicing regulation

The multi-domain splicing factor RBM5 regulates the balance between antagonistic isoforms of the apoptosis-control genes FAS/CD95, Caspase-2 and AID. An OCRE (OCtamer REpeat of aromatic residues) domain found in RBM5 is important for alternative splicing regulation and mediates interactions with components of the U4/U6.U5 tri-snRNP. We show that the RBM5 OCRE domain adopts a unique β–sheet fold. NMR and biochemical experiments demonstrate that the OCRE domain directly binds to the proline-rich C-terminal tail of the essential snRNP core proteins SmN/B/B’. The NMR structure of an OCRE-SmN peptide complex reveals a specific recognition of poly-proline helical motifs in SmN/B/B’. Mutation of conserved aromatic residues impairs binding to the Sm proteins in vitro and compromises RBM5-mediated alternative splicing regulation of FAS/CD95. Thus, RBM5 OCRE represents a poly-proline recognition domain that mediates critical interactions with the C-terminal tail of the spliceosomal SmN/B/B’ proteins in FAS/CD95 alternative splicing regulation.