Zhihua Du

Structural and biochemical insights into the dicing mechanism of mouse Dicer: A conserved lysine is critical for dsRNA cleavage

Dicer, an RNase III enzyme, initiates RNA interference by processing precursor dsRNAs into mature microRNAs and small-interfering RNAs. It is also involved in loading and activation of the RNA-induced silencing complex. Here, we report the crystal structures of a catalytically active fragment of mouse Dicer, containing the RNase IIIb and dsRNA binding domains, in its apo and Cd2+-bound forms, at 1.68- and 2.8-Å resolution, respectively. Models of this structure with dsRNA reveal that a lysine residue, highly conserved in Dicer RNase IIIa and IIIb domains and in Drosha RNase IIIb domains, has the potential to participate in the phosphodiester bond cleavage reaction by stabilizing the transition state and leaving group of the scissile bond. Mutational and enzymatic assays confirm the importance of this lysine in dsRNA cleavage, suggesting that this lysine represents a conserved catalytic residue of Dicers. The structures also reveals a ≈45-aa region within the RNase IIIb domain that harbors an α-helix at the N-terminal half and a flexible loop at the C-terminal half, features not present in previously reported structures of homologous RNase III domains from either bacterial RNase III enzymes or Giardia Dicer. N-terminal residues of this α-helix have the potential to engage in minor groove interaction with dsRNA substrates.

Crystal Structure of the First KH Domain of Human Poly(C)-binding Protein-2 in Complex with a C-rich Strand of Human Telomeric DNA at 1.7 Å

Recognition of poly(C) DNA and RNA sequences in mammalian cells is achieved by a subfamily of the KH (hnRNP K homology) domain-containing proteins known as poly(C)-binding proteins (PCBPs). To reveal the molecular basis of poly(C) sequence recognition, we have determined the crystal structure, at 1.7-Å resolution, of PCBP2 KH1 in complex with a 7-nucleotide DNA sequence (5′-AACCCTA-3′) corresponding to one repeat of the human C-rich strand telomeric DNA. The protein-DNA interaction is mediated by the combination of several stabilizing forces including hydrogen bonding, electrostatic interactions, van der Waals contacts, and shape complementarities. Specific recognition of the three cytosine residues is realized by a dense network of hydrogen bonds involving the side chains of two conserved lysines and one glutamic acid. The co-crystal structure also reveals a protein-protein dimerization interface of PCBP2 KH1 located on the opposite side of the protein from the DNA binding groove. Numerous stabilizing protein-protein interactions, including hydrophobic contacts, stacking of aromatic side chains, and a large number of hydrogen bonds, indicate that the protein-protein interaction interface is most likely genuine. Interaction of PCBP2 KH1 with the C-rich strand of human telomeric DNA suggests that PCBPs may participate in mechanisms involved in the regulation of telomere/telomerase functions.

NMR Structure of the Full-length Linear Dimer of Stem-Loop-1 RNA in the HIV-1 Dimer Initiation Site

The packaging signal of HIV-1 RNA contains a stem-loop structure, SL1, which serves as the dimerization initiation site for two identical copies of the genome and is important for packaging of the RNA genome into the budding virion and for overall infectivity. SL1 spontaneously dimerizes via a palindromic hexanucleotide sequence in its apical loop, forming a metastable kissing dimer form. Incubation with nucleocapsid protein causes this form to refold to a thermodynamically stable mature linear dimer. Here, we present an NMR structure of the latter form of the full-length SL1 sequence of the Lai HIV-1 isolate. The structure was refined using nuclear Overhauser effect and residual dipolar coupling data. The structure presents a symmetric homodimer of two RNA strands of 35 nucleotides each; it includes five stems separated by four internal loops. The central palindromic stem is surrounded by two symmetric adenine-rich 1–2 internal loops, A-bulges. All three adenines in each A-bulge are stacked inside the helix, consistent with the solution structures of shorter SL1 constructs determined previously. The outer 4-base pair stems and, proximal to them, purine-rich 1–3 internal loops, or G-bulges, are the least stable parts of the molecule. The G-bulges display high conformational variability in the refined ensemble of structures, despite the availability of many structural restraints for this region. Nevertheless, most conformations share a similar structural motif: a guanine and an adenine from opposite strands form a GA mismatch stacked on the top of the neighboring stem. The two remaining guanines are exposed, one in the minor groove and another in the major groove side of the helix, consistent with secondary structure probing data for SL1. These guanines may be recognized by the nucleocapsid protein, which binds tightly to the G-bulge in vitro.

Structure of a Construct of a Human Poly(C)-binding Protein Containing the First and Second KH Domains Reveals Insights into Its Regulatory Mechanisms

Poly(C)-binding proteins (PCBPs) are important regulatory proteins that contain three KH (hnRNP K homology) domains. Binding poly(C) D/RNA sequences via KH domains is essential for multiple PCBP functions. To reveal the basis for PCBP-D/RNA interactions and function, we determined the structure of a construct containing the first two domains (KH1-KH2) of human PCBP2 by NMR. KH1 and KH2 form an intramolecular pseudodimer. The large hydrophobic dimerization surface of each KH domain is on the side opposite the D/RNA binding interface. Chemical shift mapping indicates both domains bind poly(C) DNA motifs without disrupting the KH1-KH2 interaction. Spectral comparison of KH1-KH2, KH3, and full-length PCBP2 constructs suggests that the KH1-KH2 pseudodimer forms, but KH3 does not interact with other parts of the protein. From NMR studies and modeling, we propose possible modes of cooperative binding tandem poly(C) motifs by the KH domains. D/RNA binding may induce pseudodimer dissociation or stabilize dissociated KH1 and KH2, making protein interaction surfaces available to PCBP-binding partners. This conformational change may represent a regulatory mechanism linking D/RNA binding to PCBP functions.

Crystal structure of the third KH domain of human poly(C)-binding protein-2 in complex with a C-rich strand of human telomeric DNA at 1.6 Å resolution

KH (hnRNP K homology) domains, consisting of ∼70 amino acid residues, are present in a variety of nucleic-acid-binding proteins. Among these are poly(C)-binding proteins (PCBPs), which are important regulators of mRNA stability and posttranscriptional regulation in general. All PCBPs contain three different KH domains and recognize poly(C)-sequences with high affinity and specificity. To reveal the molecular basis of poly(C)-sequence recognition, we have determined the crystal structure, at 1.6 Å resolution, of PCBP2 KH3 domain in complex with a 7-nt DNA sequence (5′-AACCCTA-3′) corresponding to one repeat of the C-rich strand of human telomeric DNA. The domain assumes a type-I KH fold in a βααββα configuration. The protein–DNA interface could be studied in unprecedented detail and is made up of a series of direct and water-mediated hydrogen bonds between the protein and the DNA, revealing an especially dense network involving several structural water molecules for the last 2 nt in the core recognition sequence. Unlike published KH domain structures, the protein crystallizes without protein–protein contacts, yielding new insights into the dimerization properties of different KH domains. A nucleotide platform, an interesting feature found in some RNA molecules, was identified, evidently for the first time in DNA.

Comparative studies of frameshifting and nonframeshifting RNA pseudoknots: A mutational and NMR investigation of pseudoknots derived from the bacteriophage T2 gene 32 mRNA and the retroviral gag-pro frameshift site

Mutational and NMR methods were used to investigate features of sequence, structure, and dynamics that are associated with the ability of a pseudoknot to stimulate a -1 frameshift. In vitro frameshift assays were performed on retroviral gag-pro frameshift-stimulating pseudoknots and their derivatives, a pseudoknot from the gene 32 mRNA of bacteriophage T2 that is not naturally associated with frameshifting, and hybrids of these pseudoknots. Results show that the gag-pro pseudoknot from human endogenous retrovirus-K10 (HERV) stimulates a -1 frameshift with an efficiency similar to that of the closely related retrovirus MMTV. The bacteriophage T2 mRNA pseudoknot was found to be a poor stimulator of frameshifting, supporting a hypothesis that the retroviral pseudoknots have distinctive properties that make them efficient frameshift stimulators. A hybrid, designed by combining features of the bacteriophage and retroviral pseudoknots, was found to stimulate frameshifting while retaining significant structural similarity to the nonframeshifting bacteriophage pseudoknot. Mutational analyses of the retroviral and hybrid pseudoknots were used to evaluate the effects of an unpaired (wedged) adenosine at the junction of the pseudoknot stems, changing the base pairs near the junction of the two stems, and changing the identity of the loop 2 nucleotide nearest the junction of the stems. Pseudoknots both with and without the wedged adenosine can stimulate frameshifting, though the identities of the nucleotides near the stem1/stem2 junction do influence efficiency. NMR data showed that the bacteriophage and hybrid pseudoknots are similar in their local structure at the junction of the stems, indicating that pseudoknots that are similar in this structural feature can differ radically in their ability to stimulate frameshifting. NMR methods were used to compare the internal motions of the bacteriophage T2 pseudoknot and representative frameshifting pseudoknots. The stems of the investigated pseudoknots are similarly well ordered on the time scales to which nitrogen-15 relaxation data are sensitive; however, solvent exchange rates for protons at the junction of the two stems of the nonframeshifting bacteriophage pseudoknot are significantly slower than the analogous protons in the representative frameshifting pseudoknots.

Highly conserved RNA pseudoknots at the gag-pol junction of HIV-1 suggest a novel mechanism of −1 ribosomal frameshifting

-1 programmed ribosomal frameshifting (PRF) is utilized by many viruses to synthesize their enzymatic (Pol) and structural (Gag) proteins at a defined ratio. For efficient -1 PRF, two cis-acting elements are required: a heptanucleotide frameshift site and a downstream stimulator such as a pseudoknot. We have analyzed the gag-pol junction sequences from 4254 HIV-1 strains. Approximately ninety-five percent of the sequences can form four pseudoknots PK1-PK4 (∼ 97% contain PK1, PK3, and PK4), covering ∼ 72 nt including the frameshift site. Some pseudoknots are mutually excluded due to sequence overlap. PK1 and PK3 arrange tandemly. Their stems form a quasi-continuous helix of ∼ 22 bp. We propose a novel mechanism for possible roles of these pseudoknots. Multiple alternative structures may exist at the gag-pol junction. In most strains, the PK1-PK3 tandem pseudoknots may dominate the structurally heterogeneous pool of RNA due to their greater overall stability. The tandem pseudoknots may function as a breaking system to slow down the ribosome. The ribosome unwinds PK1 and stem 1 of PK3 before it can reach the frameshift site. Then, PK4 can form rapidly because the intact stem 2 of PK3 makes up a large part of the stem 1 of PK4. The newly formed PK4 jams the entrance of the mRNA tunnel. The process then proceeds as in a typical case of -1 PRF. This mechanism incorporates several exquisite new features while still being consistent with the current paradigm of pseudoknot-dependent -1 PRF.

The structure of full-length human CTNNBL1 reveals a distinct member of the armadillo-repeat protein family

Catenin-β-like protein 1 (CTNNBL1) is a highly conserved protein with multiple functions, one of which is to act as an interaction partner of the antibody-diversification enzyme activation-induced cytidine deaminase (AID) for its nuclear import and subnuclear trafficking. Here, the crystal structure of full-length human CTNNBL1 is reported. The protein contains six armadillo (ARM) repeats that pack into a superhelical ARM domain. This ARM domain is unique within the ARM protein family owing to the presence of several unusual structural features. Moreover, CTNNBL1 contains significant and novel non-ARM structures flanking both ends of the central ARM domain. A strong continuous hydrophobic core runs through the whole structure, indicating that the ARM and non-ARM structures fold together to form an integral structure. This structure defines a highly restrictive and discriminatory protein-binding groove that is not observed in other ARM proteins. The presence of a cluster of histidine residues in the groove implies a pH-sensitive histidine-mediated mechanism that may regulate protein binding activity. The many unique structural features of CTNNBL1 establish it as a distinct member of the ARM protein family. The structure provides critical insights into the molecular interactions between CTNNBL1 and its protein partners, especially AID.

Structure of the FP domain of Fbxo7 reveals a novel mode of protein-protein interaction

The FP (Fbxo7/PI31) domains found in the F-box protein Fbxo7 and the proteasome inhibitor PI31 mediate the homodimerization and heterodimerization of Fbxo7 and PI31. Fbxo7 is the substrate-recognition subunit of the SCF(Fbxo7) (Skp1-Cul1-F-box protein) E3 ubiquitin ligase that catalyzes the ubiquitination of hepatoma up-regulated protein (HURP) and inhibitor of apoptosis protein (IAP). Fbxo7 also interacts with proteins that are not substrates of the ubiquitin proteasome system, such as Cdk6 and PI31. Here, the crystal structure of the Fbxo7 FP domain is reported at 2.0 Å resolution. The Fbxo7 FP domain adopts an α/β-fold similar to that of the PI31 FP domain. However, an α-helix and three β-strands in the Fbxo7 FP domain are longer than their counterparts in the PI31 FP domain. The differences in these secondary-structural elements are spatially clustered to define a more structured and extended C-terminal end of the Fbxo7 FP domain. The two FP domains also differ substantially in the length and conformation of the longest connecting loop. More importantly, structural differences between the two FP domains lead to drastically different modes of inter-domain protein-protein interaction. The inter-domain interface of the Fbxo7 FP domain is defined by the α-helical surface in one protomer and the β-sheet surface in the other protomer, whereas for the PI31 domain it is defined by either the α-helical surfaces or the β-sheet surfaces in both protomers. The inter-domain interaction of the Fbxo7 FP domain is much more extensive, featuring a larger contact surface area, better shape complementarity and more hydrophobic and hydrogen-bonding interactions. The Fbxo7 FP domain also has the potential to bind two protein partners simultaneously using the α-helical and β-sheet surfaces. The results of this structural study provide critical insights into how Fbxo7 may dimerize (or multimerize) and interact with other regulatory proteins via the FP domain.

Possible involvement of coaxially stacked double pseudoknots in the regulation of −1 programmed ribosomal frameshifting in RNA viruses

−1 programmed ribosomal frameshifting (PRF) in viruses is often stimulated by a pseudoknot downstream from the slippery sequence. At the PRF junction of HIV-1, transmissible gastroenteritis virus (TGEV), Barmah Forest virus (BFV), Fort Morgan virus (FMV), and Equine arteritis virus (EAV), we identified potential double pseudoknots in either a tandem mode or embedded mode. In viruses with tandem pseudoknots (5′PK & 3′PK), the slippery sequence is encompassed in the 5′PK. The ribosome needs to unwind the 5′PK to get to the slippery sequence. In HIV-1, the 3′PK and several alternative structures are mutually exclusive. Disruption of the tandem pseudoknots may enable one of the alternative structures to form as the effective frameshift stimulator. In TGEV/BFV/FMV, the 3′PK is a conventional frameshift stimulator. In all cases, the tandem pseudoknots may slow down the ribosome before it reaches the conventional PRF signals. In EAV, a compact pseudoknot is embedded within loop2 of the otherwise conventional frameshift-stimulating pseudoknot. All double pseudoknots have the potential to stack their stems coaxially. We built structural models of the HIV-1 and EAV double pseudoknots to show that both the tandem and embedded modes are feasible and reasonable. We hypothesize that the fundamental reason for the viruses to utilize coaxially stacked double pseudoknots is to increase the overall stability of the frameshift regulating structure, and avoid an ultra-stable single pseudoknot which may become a ribosomal roadblock. Our results significantly expand the repertoire of RNA structures and dynamics that may potentially involve in −1 PRF regulation.