Seth Chitayat

Structural basis of CBP/p300 recruitment in leukemia induction by E2A-PBX1

E-proteins are critical transcription factors in B-cell lymphopoiesis. E2A, 1 of 3 E-protein-encoding genes, is implicated in the induction of acute lymphoblastic leukemia through its involvement in the chromosomal translocation 1;19 and consequent expression of the E2A-PBX1 oncoprotein. An interaction involving a region within the N-terminal transcriptional activation domain of E2A-PBX1, termed the PCET motif, which has previously been implicated in E-protein silencing, and the KIX domain of the transcriptional coactivator CBP/p300, critical for leukemogenesis. However, the structural details of this interaction remain unknown. Here we report the structure of a 1:1 complex between PCET motif peptide and the KIX domain. Residues throughout the helical PCET motif that contact the KIX domain are important for both binding KIX and bone marrow immortalization by E2A-PBX1. These results provide molecular insights into E-protein-driven differentiation of B-cells and the mechanism of E-protein silencing, and reveal the PCET/KIX interaction as a therapeutic target for E2A-PBX1-induced leukemia.

Atypical Cohesin-Dockerin Complex Responsible for Cell Surface Attachment of Cellulosomal Components BINDING FIDELITY, PROMISCUITY, AND STRUCTURAL BUTTRESSES

Background: The type IIIe CohE-XDoc interaction connects cellulosomal components to the cell wall. Results: The dockerin structure in the CohE-XDoc complex exhibits an atypical calcium-binding loop disrupted by a 13-residue insert. Conclusion: The dockerin inserts evolved to serve as novel structural buttresses that support the stalklike X-module conformation. Significance: The type IIIe CohE-XDoc complex underscores dockerin divergence and provides insight into the determinants for cohesin-dockerin specificity. The rumen bacterium Ruminococcus flavefaciens produces a highly organized multienzyme cellulosome complex that plays a key role in the degradation of plant cell wall polysaccharides, notably cellulose. The R. flavefaciens cellulosomal system is anchored to the bacterial cell wall through a relatively small ScaE scaffoldin subunit, which bears a single type IIIe cohesin responsible for the attachment of two major dockerin-containing scaffoldin proteins, ScaB and the cellulose-binding protein CttA. Although ScaB recruits the catalytic machinery onto the complex, CttA mediates attachment of the bacterial substrate via its two putative carbohydrate-binding modules. In an effort to understand the structural basis for assembly and cell surface attachment of the cellulosome in R. flavefaciens, we determined the crystal structure of the high affinity complex (Kd = 20.83 nm) between the cohesin module of ScaE (CohE) and its cognate X-dockerin (XDoc) modular dyad from CttA at 1.97-Å resolution. The structure reveals an atypical calcium-binding loop containing a 13-residue insert. The results further pinpoint two charged specificity-related residues on the surface of the cohesin module that are responsible for specific versus promiscuous cross-strain binding of the dockerin module. In addition, a combined functional role for the three enigmatic dockerin inserts was established whereby these extraneous segments serve as structural buttresses that reinforce the stalklike conformation of the X-module, thus segregating its tethered complement of cellulosomal components from the cell surface. The novel structure of the RfCohE-XDoc complex sheds light on divergent dockerin structure and function and provides insight into the specificity features of the type IIIe cohesin-dockerin interaction.

Identification and Characterization of an 8-kDa Light Chain Associated with Dictyostelium discoideum MyoB, a Class I Myosin

Dictyostelium discoideum MyoB is a single-headed class I myosin. Analysis of purified MyoB by SDS-PAGE indicated the presence of an ∼9-kDa light chain. A tryptic digest of MyoB yielded a partial sequence for the light chain that exactly matched a sequence in a 73-amino acid, 8,296-Da protein (dictyBase number DDB0188713). This protein, termed MlcB, contains two EF-hand motifs and shares ∼30% sequence identity with the N- and C-terminal lobes of calmodulin. FLAG-MlcB expressed in Dictyostelium co-immunoprecipitated with MyoB but not with the related class myosins and MyoD. Recombinant MlcB bound Ca2+ with a Kd value of 0.2 μm and underwent a Ca2+-induced change in conformation that increased α-helical content and surface hydrophobicity. Mutational analysis showed that the first EF-hand was responsible for Ca2+ binding. In the presence and absence of Ca2+ MlcB was a monomer in solution and bound to a MyoB IQ motif peptide with a Kd value of ∼0.5 μm. A MyoB head-neck construct with a Ser to Glu mutation at the TEDS site bound MlcB and displayed an actin-activated Mg2+ ATPase activity that was insensitive to Ca2+. We conclude that MlcB represents a novel type of small myosin light chain that binds to IQ motifs in a manner comparable with a single lobe of a typical four-EF-hand protein.

The Solution Structure of the C-terminal Modular Pair from Clostridium perfringens μ-Toxin Reveals a Noncellulosomal Dockerin Module

The genome of the opportunistic pathogen Clostridium perfringens encodes a large number of secreted glycoside hydrolases. Their predicted activities indicate that they are involved in the breakdown of complex carbohydrates and other glycans found in the mucosal layer of the human gastrointestinal tract, within the extracellular matrix, and on the surface of host cells. One such group of these enzymes is the family 84 glycoside hydrolases, which has predicted hyaluronidase activity and comprises five members [C. perfringens glycoside hydrolase family 84 (CpGH84) A-E]. The first identified member, CpGH84A, corresponds to the mu-toxin whose modular architecture includes an N-terminal catalytic domain, four family 32 carbohydrate-binding modules, three FIVAR modules of unknown function, and a C-terminal putative calcium-binding module. Here, we report the solution NMR structure of the C-terminal modular pair from the mu-toxin. The three-helix bundle FIVAR module displays structural homology to a heparin-binding module within the N-terminal of the a C protein from group B Streptoccocus. The C-terminal module has a typical calcium-binding dockerin fold comprising two anti-parallel helices that form a planar face with EF-hand calcium-binding loops at opposite ends of the module. The size of the helical face of the mu-toxin dockerin module is approximately equal to the planar region recently identified on the surface of a cohesin-like X82 module of CpGH84C. Size-exclusion chromatography and heteronuclear NMR-based chemical shift mapping studies indicate that the helical face of the dockerin module recognizes the CpGH84C X82 module. These studies represent the structural characterization of a noncellulolytic dockerin module and its interaction with a cohesin-like X82 module. Dockerin/X82-mediated enzyme complexes may have important implications in the pathogenic properties of C. perfringens.

An unusual mode of galactose recognition by a family 32 carbohydrate-binding module.

Carbohydrate-binding modules (CBMs) are ancillary modules commonly associated with carbohydrate-active enzymes (CAZymes) that function to mediate the adherence of the parent enzyme to its carbohydrate substrates. CBM family 32 (CBM32) is one of the most diverse CBM families, whose members are commonly found in bacterial CAZymes that modify eukaryotic glycans. One such example is the putative μ-toxin, CpGH84A, of the family 84 glycoside hydrolases, which comprises an N-terminal putative β-N-acetylglucosaminidase catalytic module and four tandem CBM32s. Here, we report a unique mode of galactose recognition by the first CBM32, CBM32-1 from CpGH84A. Solution NMR-based analyses of CpGH84A CBM32-1 indicate a divergent subset of residues, located in ordered loops at the apex of the CBM, conferring specificity for the galacto-configured sugars galactose, GalNAc, and LacNAc that differs from those of the canonical galactose-binding CBM32s. This study showcases the impressive variability in ligand binding by this CBM family and offers insight into the growing role of these modules in the interaction of CAZymes with eukaryotic glycans.

Intramolecular clasp of the cellulosomal Ruminococcus flavefaciens ScaA dockerin module confers structural stability

The cellulosome is a large extracellular multi‐enzyme complex that facilitates the efficient hydrolysis and degradation of crystalline cellulosic substrates. During the course of our studies on the cellulosome of the rumen bacterium Ruminococcus flavefaciens, we focused on the critical ScaA dockerin (ScaADoc), the unique dockerin that incorporates the primary enzyme‐integrating ScaA scaffoldin into the cohesin‐bearing ScaB adaptor scaffoldin. In the absence of a high‐resolution structure of the ScaADoc module, we generated a computational model, and, upon its analysis, we were surprised to discover a putative stacking interaction between an N‐terminal Trp and a C‐terminal Pro, which we termed intramolecular clasp. In order to verify the existence of such an interaction, these residues were mutated to alanine. Circular dichroism spectroscopy, intrinsic tryptophan and ANS fluorescence, and NMR spectroscopy indicated that mutation of these residues has a destabilizing effect on the functional integrity of the Ca2+‐bound form of ScaADoc. Analysis of recently determined dockerin structures from other species revealed the presence of other well‐defined intramolecular clasps, which consist of different types of interactions between selected residues at the dockerin termini. We propose that this thematic interaction may represent a major distinctive structural feature of the dockerin module.

Mapping acetylation sites in E2A identifies a conserved lysine residue in activation domain 1 that promotes CBP/p300 recruitment and transcriptional activation.

E-proteins are basic helix-loop-helix transcription factors that function in cell type specification. The gene E2A encodes two E-proteins, E12 and E47, which are required in B-lymphopoiesis. E2A proteins can interact directly with the transcriptional co-activators and lysine acetyltranferases (KATs) CBP, p300 and PCAF to induce target gene transcription. Prior investigations have shown that the E2A-encoded isoform E2-5 is acetylated by CBP, p300 or PCAF in vitro or in vivo. However, E2-5 lacks the important N-terminal activation domain AD1. Furthermore, the acetylated residues in E-proteins have not been mapped, and the functional consequences of acetylation are largely unknown. Here, we use mutagenesis to show that a lysine residue at position 34 within AD1 of E12/E47 is acetylated by CBP/p300 and PCAF. Lys34 lies adjacent to a conserved helical LXXLL motif that interacts directly with the KIX domain of CBP/p300. We show that acetylation at Lys34 increases the affinity of AD1 for the KIX domain and enhances AD1-driven transcriptional induction. Our results illustrate for the first time that AD1 can both recruit, and be acetylated by, KATs and that KAT recruitment may promote transcriptional induction in part through acetylation of AD1 itself.

Conformational analysis of StrH, the surface-attached exo-β-D-N-acetylglucosaminidase from Streptococcus pneumoniae.

Streptococcus pneumoniae is a serious human pathogen that presents on its surface numerous proteins involved in the host-bacterium interaction. The carbohydrate-active enzymes are particularly well represented among these surface proteins, and many of these are known virulence factors, highlighting the importance of carbohydrate processing by this pathogen. StrH is a surface-attached exo-β-D-N-acetylglucosaminidase that cooperates with the sialidase NanA and the β-galactosidase BgaA to sequentially degrade the nonreducing terminal arms of complex N-linked glycans. This enzyme is a large multi-modular protein that is notable for its tandem N-terminal family GH20 catalytic modules, whose individual X-ray crystal structures were recently reported. StrH also contains C-terminal tandem G5 modules, which are uncharacterized. Here, we report the NMR-determined solution structure of the first G5 module in the tandem, G5-1, which along with the X-ray crystal structures of the GH20 modules was used in conjunction with small-angle X-ray scattering to construct a pseudo-atomic model of full-length StrH. The results reveal a model in which StrH adopts an elongated conformation that may project the catalytic modules away from the surface of the bacterium to a distance of up to ~250 Å.

Structural and functional characterization of a novel type‐III dockerin from Ruminococcus flavefaciens

ScaB cohesin binds to ScaADoc by enzyme linked immunosorbent assay (View interaction)

1H, 15N and 13C backbone and side-chain resonance assignments of a family 32 carbohydrate-binding module from the Clostridium perfringens NagH

The Gram-positive anaerobe Clostridium perfringens is an opportunistic bacterial pathogen that secretes a battery of enzymes involved in glycan degradation. These glycoside hydrolases are thought to be involved in turnover of mucosal layer glycans, and in the spread of major toxins commonly associated with the development of gastrointestinal diseases and gas gangrene in humans. These enzymes employ multi-modularity and carbohydrate-binding function to degrade extracellular eukaryotic host sugars. Here, we report the full 1H, 15N and 13C chemical shift resonance assignments of the first family 32 carbohydrate-binding module from NagH, a secreted family 84 glycoside hydrolase.