Rodolfo M. Rasia

Hadamard Amino-Acid-Type Edited NMR Experiment for Fast Protein Resonance Assignment

An original Hadamard-encoding scheme allows discrimination among seven amino acid types in a single two-dimensional NMR experiment. Combined with hyperdimensional NMR techniques, this presents a promising new method for fast, automated backbone resonance assignment of proteins in only a few hours time.

Structural Determinants of Substrate Binding to Bacillus cereus Metallo-β-lactamase

Binding and hydrolysis of the β-lactams cefotaxime, cephapirin, imipenem, and benzylpenicillin by the metallo-β-lactamase from Bacillus cereus were studied by presteady state kinetic measurements. In all cases, the substrate was unmodified in the most populated reaction intermediate, and no chemically modified substrate species accumulated to a detectable amount. The cephalosporins tested showed similar formation rate constants for this intermediate, and they differed mostly in their decay rates. Formation of a non-productive enzyme ·substrate complex was detected for imipenem. The substrate binding differences can be accounted for by considering the structural features of each substrate. The apoenzyme could not bind any of the substrates, but binding was restored when the apoenzyme was reconstituted with Zn(II), revealing that the metal ions are the main determinants of substrate binding. This evidence is in line with the lack of an optimized substrate recognition patch in B1 and B3 metallo-β-lactamases that provides a broad substrate spectrum.

Structure and RNA interactions of the plant microRNA processing-associated protein HYL1.

HYL1 is a double-stranded RNA binding protein involved in microRNA processing in plants. HYL1 enhances the efficiency and precision of the RNase III protein DCL1 and participates in microRNA strand selection. In this work, we dissect the contributions of the domains of HYL1 to the binding of RNA targets. We found that the first domain is the main contributor to RNA binding. Mapping of the interaction regions by nuclear magnetic resonance on the structure of HYL1 RNA-binding domains showed that the difference in binding capabilities can be traced to sequence divergence in β2-β3 loop. The possible role of each domain is discussed in light of previous experimental data.

Selective isotopic unlabeling of proteins using metabolic precursors: application to NMR assignment of intrinsically disordered proteins.

Selective isotopic unlabeling of proteins can provide important residue‐type information as well as reduce congestion of NMR spectra. However, metabolic scrambling often complicates the final isotope‐labeling pattern. Here, an array of metabolic precursors is used to perform robust, residue‐specific unlabeling of proteins. The resulting isotopic‐labeling patterns are predictable and nicely complement NMR experiments that differentiate residue types. This approach has widespread applications, but it is particularly relevant for proteins that lack sequence complexity or a defined tertiary structure.

Using Genetically Encodable Self-Assembling Gd(III) Spin Labels To Make In-Cell Nanometric Distance Measurements.

Double electron-electron resonance (DEER) can be used to study the structure of a protein in its native cellular environment. Until now, this has required isolation, in vitro labeling, and reintroduction of the protein back into the cells. We describe a completely biosynthetic approach that avoids these steps. It exploits genetically encodable lanthanide-binding tags (LBT) to form self-assembling Gd(III) metal-based spin labels and enables direct in-cell measurements. This approach is demonstrated using a pair of LBTs encoded one at each end of a 3-helix bundle expressed in E. coli grown on Gd(III) -supplemented medium. DEER measurements directly on these cells produced readily detectable time traces from which the distance between the Gd(III) labels could be determined. This work is the first to use biosynthetically produced self-assembling metal-containing spin labels for non-disruptive in-cell structural measurements.

Human initiation factor eIF3 subunit b interacts with HCV IRES RNA through its N-terminal RNA recognition motif

Many viral mRNAs contain a 5′‐UTR RNA element called internal ribosome‐entry site (IRES), which bypasses the requirement of some canonical initiation factors allowing cap‐independent translation. The IRES of hepatitis‐C virus drives translation by directly recruiting 40S ribosomal subunits and binds to eIF3 which plays a critical role in both cap‐dependent and cap‐independent translation. However, the molecular basis for eIF3 activity in either case remains enigmatic. Here we report that subunit b of the eIF3 complex directly binds to HCV IRES domain III via its N‐terminal‐RRM. Because eIF3b was previously shown to be involved in eIF3j binding, biological implications are discussed.

The Use of Mn(II) Bound to His-tags as Genetically Encodable Spin-Label for Nanometric Distance Determination in Proteins.

A genetically encodable paramagnetic spin-label capable of self-assembly from naturally available components would offer a means for studying the in-cell structure and interactions of a protein by electron paramagnetic resonance (EPR). Here, we demonstrate pulse electron-electron double resonance (DEER) measurements on spin-labels consisting of Mn(II) ions coordinated to a sequence of histidines, so-called His-tags, that are ubiquitously added by genetic engineering to facilitate protein purification. Although the affinity of His-tags for Mn(II) was low (800 μM), Mn(II)-bound His-tags yielded readily detectable DEER time traces even at concentrations expected in cells. We were able to determine accurately the distance between two His-tag Mn(II) spin-labels at the ends of a rigid helical polyproline peptide of known structure, as well as at the ends of a completely cell-synthesized 3-helix bundle. This approach not only greatly simplifies the labeling procedure but also represents a first step towards using self-assembling metal spin-labels for in-cell distance measurements.

Protein Signatures That Promote Operator Selectivity among Paralog MerR Monovalent Metal Ion Regulators

Background: Two nucleotide bases distinguish promoters controlled by paralog MerR monovalent metalloregulators, avoiding cross-activation. Results: Specific residues within the DNA-binding region of the regulators were identified as responsible for the selectivity in the operator recognition. Conclusion: Co-evolution of both the regulator and its target operator sequences prevents cross-activation of paralog regulatory circuits. Significance: The basis for regulator/operator specificity among MerR monovalent metalloregulators is described. Two paralog transcriptional regulators of the MerR family, CueR and GolS, are responsible for monovalent metal ion sensing and resistance in Salmonella enterica. Although similar in sequence and also in their target binding sites, these proteins differ in signal detection and in the set of target genes they control. Recently, we demonstrated that selective promoter recognition depends on the presence of specific bases located at positions 3′ and 3 within the operators they interact with. Here, we identify the amino acid residues within the N-terminal DNA-binding domain of these sensor proteins that are directly involved in operator discrimination. We demonstrate that a methionine residue at position 16 of GolS, absolutely conserved among GolS-like proteins but absent in all CueR-like xenologs, is the key to selectively recognize operators that harbor the distinctive GolS-operator signature, whereas the residue at position 19 finely tunes the regulator/operator interaction. Furthermore, swapping these residues switches the set of genes recognized by these transcription factors. These results indicate that co-evolution of a regulator and its cognate operators within the bacterial cell provides the conditions to avoid cross-recognition and guarantees the proper response to metal injury.

Second double-stranded RNA binding domain of dicer-like ribonuclease 1: structural and biochemical characterization.

Dicer-like ribonuclease III enzymes are involved in different paths related to RNA silencing in plants. Little is known about the structural aspects of these processes. Here we present a structural characterization of the second double-stranded RNA binding domain (dsRBD) of DCL1, which is presumed to participate in pri-micro-RNA recognition and subcellular localization of this protein. We determined the solution structure and found that it has a canonical fold but bears some variation with respect to other homologous domains. We also found that this domain binds both double-stranded RNA and double-stranded DNA, in contrast to most dsRBDs. Our characterization shows that this domain likely has functions other than substrate recognition and binding.

Induced folding in RNA recognition by Arabidopsis thaliana DCL1

DCL1 is the ribonuclease that carries out miRNA biogenesis in plants. The enzyme has two tandem double stranded RNA binding domains (dsRBDs) in its C-terminus. Here we show that the first of these domains binds precursor RNA fragments when isolated and cooperates with the second domain in the recognition of substrate RNA. Remarkably, despite showing RNA binding activity, this domain is intrinsically disordered. We found that it acquires a folded conformation when bound to its substrate, being the first report of a complete dsRBD folding upon binding. The free unfolded form shows tendency to adopt folded conformations, and goes through an unfolded bound state prior to the folding event. The significance of these results is discussed by comparison with the behavior of other dsRBDs.