Tyson R. Shepherd

A novel pathway spatiotemporally activates Rac1 and redox signaling in response to fluid shear stress

The GEF Tiam1 acts as a novel molecular link to the VE-cadherin–p67phox–Par3 polarity complex, leading to localized activation of Rac1 and NADPH oxidase in response to fluid flow.

Distinct Ligand Specificity of the Tiam1 and Tiam2 PDZ Domains

Guanine nucleotide exchange factor proteins of the Tiam family are activators of the Rho GTPase Rac1 and critical for cell morphology, adhesion, migration, and polarity. These proteins are modular and contain a variety of interaction domains, including a single post-synaptic density-95/discs large/zonula occludens-1 (PDZ) domain. Previous studies suggest that the specificities of the Tiam1 and Tiam2 PDZ domains are distinct. Here, we sought to conclusively define these specificities and determine their molecular origin. Using a combinatorial peptide library, we identified a consensus binding sequence for each PDZ domain. Analysis of these consensus sequences and binding assays with peptides derived from native proteins indicated that these two PDZ domains have overlapping but distinct specificities. We also identified residues in two regions (S(0) and S(-2) pockets) of the Tiam1 PDZ domain that are important determinants of ligand specificity. Site-directed mutagenesis of four nonconserved residues in these two regions along with peptide binding analyses confirmed that these residues are crucial for ligand affinity and specificity. Furthermore, double mutant cycle analysis of each region revealed energetic couplings that were dependent on the ligand being investigated. Remarkably, a Tiam1 PDZ domain quadruple mutant had the same specificity as the Tiam2 PDZ domain. Finally, analysis of Tiam family PDZ domain sequences indicated that the PDZ domains segregate into four distinct families based on the residues studied here. Collectively, our data suggest that Tiam family proteins have highly evolved PDZ domain-ligand interfaces with distinct specificities and that they have disparate PDZ domain-dependent biological functions.

The structure of the Tiam1 PDZ domain/ phospho-syndecan1 complex reveals a ligand conformation that modulates protein dynamics.

PDZ (PSD-95/Dlg/ZO-1) domains are protein-protein interaction modules often regulated by ligand phosphorylation. Here, we investigated the specificity, structure, and dynamics of Tiam1 PDZ domain/ligand interactions. We show that the PDZ domain specifically binds syndecan1 (SDC1), phosphorylated SDC1 (pSDC1), and SDC3 but not other syndecan isoforms. The crystal structure of the PDZ/SDC1 complex indicates that syndecan affinity is derived from amino acids beyond the four C-terminal residues. Remarkably, the crystal structure of the PDZ/pSDC1 complex reveals a binding pocket that accommodates the phosphoryl group. Methyl relaxation experiments of PDZ/SCD1 and PDZ/pSDC1 complexes reveal that PDZ-phosphoryl interactions dampen dynamic motions in a distal region of the PDZ domain by decoupling them from the ligand-binding site. Our data are consistent with a selection model by which specificity and phosphorylation regulate PDZ/syndecan interactions and signaling events. Importantly, our relaxation data demonstrate that PDZ/phospho-ligand interactions regulate protein dynamics and their coupling to distal sites.

Crystal structure of RlmM, the 2′O-ribose methyltransferase for C2498 of Escherichia coli 23S rRNA

RlmM (YgdE) catalyzes the S-adenosyl methionine (AdoMet)-dependent 2′O methylation of C2498 in 23S ribosomal RNA (rRNA) of Escherichia coli. Previous experiments have shown that RlmM is active on 23S rRNA from an RlmM knockout strain but not on mature 50S subunits from the same strain. Here, we demonstrate RlmM methyltransferase (MTase) activity on in vitro transcribed 23S rRNA and its domain V. We have solved crystal structures of E. coli RlmM at 1.9 Å resolution and of an RlmM–AdoMet complex at 2.6 Å resolution. RlmM consists of an N-terminal THUMP domain and a C-terminal catalytic Rossmann-like fold MTase domain in a novel arrangement. The catalytic domain of RlmM is closely related to YiiB, TlyA and fibrillarins, with the second K of the catalytic tetrad KDKE shifted by two residues at the C-terminal end of a beta strand compared with most 2′O MTases. The AdoMet-binding site is open and shallow, suggesting that RNA substrate binding may be required to form a conformation needed for catalysis. A continuous surface of conserved positive charge indicates that RlmM uses one side of the two domains and the inter-domain linker to recognize its RNA substrate.

Structural and functional insights into the molecular mechanism of rRNA m6A methyltransferase RlmJ

RlmJ catalyzes the m6A2030 methylation of 23S rRNA during ribosome biogenesis in Escherichia coli. Here, we present crystal structures of RlmJ in apo form, in complex with the cofactor S-adenosyl-methionine and in complex with S-adenosyl-homocysteine plus the substrate analogue adenosine monophosphate (AMP). RlmJ displays a variant of the Rossmann-like methyltransferase (MTase) fold with an inserted helical subdomain. Binding of cofactor and substrate induces a large shift of the N-terminal motif X tail to make it cover the cofactor binding site and trigger active-site changes in motifs IV and VIII. Adenosine monophosphate binds in a partly accommodated state with the target N6 atom 7 Å away from the sulphur of AdoHcy. The active site of RlmJ with motif IV sequence 164DPPY167 is more similar to DNA m6A MTases than to RNA m62A MTases, and structural comparison suggests that RlmJ binds its substrate base similarly to DNA MTases T4Dam and M.TaqI. RlmJ methylates in vitro transcribed 23S rRNA, as well as a minimal substrate corresponding to helix 72, demonstrating independence of previous modifications and tertiary interactions in the RNA substrate. RlmJ displays specificity for adenosine, and mutagenesis experiments demonstrate the critical roles of residues Y4, H6, K18 and D164 in methyl transfer.

De novo design and synthesis of a 30-cistron translation-factor module

Abstract Two of the many goals of synthetic biology are synthesizing large biochemical systems and simplifying their assembly. While several genes have been assembled together by modular idempotent cloning, it is unclear if such simplified strategies scale to very large constructs for expression and purification of whole pathways. Here we synthesize from oligodeoxyribonucleotides a completely de-novo-designed, 58-kb multigene DNA. This BioBrick plasmid insert encodes 30 of the 31 translation factors of the PURE translation system, each His-tagged and in separate transcription cistrons. Dividing the insert between three high-copy expression plasmids enables the bulk purification of the aminoacyl-tRNA synthetases and translation factors necessary for affordable, scalable reconstitution of an in vitro transcription and translation system, PURE 3.0.

Enzymatic synthesis of gene-length single-stranded DNA

Single-stranded DNA (ssDNA) increases the likelihood of homology directed repair with reduced cellular toxic ity, yet ssDNA synthesis strategies are limited by the maximum length attainable, as well control over nucleotide composition. Here, we apply purely enzymatic synthesis to generate ssDNA greater than 15 kb using asymmetric PCR, and illustrate the incorporation of diverse modified nucleotides for therapeutic and imaging applications.

Refactoring gene sequences for broad assembly standards compatibility

Four cloning standards in synthetic biology are BioBrick, BglBrick, MoClo and GoldenBraid, with each requiring their constitutive parts be compatible with the associated restriction enzymes. To standardize parts for the broadest usage, it would be useful to synthesize genes that are simultaneously compatible with all 4 popular assembly strategies. Here it is shown that using a defined set of rules, implemented in a computational program, any protein coding sequence can be made compatible with all four standards by silent mutations. Using a coding sequence as an input, all BioBrick, BglBrick, MoClo, and GoldenBraid restriction sites and chi recombination hot spots can be destroyed with silent mutations that approximate the codon usage of the organism. As an application, all open reading frames in the model organisms Escherichia Coli and Bacillus Subtilis are computationally refactored, showing the feasibility of implementing this umbrella strategy for synthesizing genes with the broadest compatibility.