Hydration dynamics of hyaluronan and dextran

Hyaluronan is a polysaccharide, which is ubiquitous in vertebrates and has been reported to be strongly hydrated in a biological environment. We study the hydration of hyaluronan in solution using the rotational dynamics of water as a probe. We measure these dynamics with polarization-resolved femtosecond-infrared and terahertz time-domain spectroscopies. Both experiments reveal that a subensemble of water molecules is slowed down in aqueous solutions of hyaluronan amounting to ～15 water molecules per disaccharide unit. This quantity is consistent with what would be expected for the first hydration shell. Comparison of these results to the water dynamics in aqueous dextran solution, a structurally similar polysaccharide, yields remarkably similar results. This suggests that the observed interaction with water is a common feature for hydrophilic polysaccharides and is not specific to hyaluronan.     

Crystal Structure of the Formin mDia1 in Autoinhibited Conformation

Background

Formin proteins utilize a conserved formin homology 2 (FH2) domain to nucleate new actin filaments. In mammalian diaphanous-related formins (DRFs) the FH2 domain is inhibited through an unknown mechanism by intramolecular binding of the diaphanous autoinhibitory domain (DAD) and the diaphanous inhibitory domain (DID).

Methodology/Principal Findings

Here we report the crystal structure of a complex between DID and FH2-DAD fragments of the mammalian DRF, mDia1 (mammalian diaphanous 1 also called Drf1 or p140mDia). The structure shows a tetrameric configuration (4 FH2 + 4 DID) in which the actin-binding sites on the FH2 domain are sterically occluded. However biochemical data suggest the full-length mDia1 is a dimer in solution (2 FH2 + 2 DID). Based on the crystal structure, we have generated possible dimer models and found that architectures of all of these models are incompatible with binding to actin filament but not to actin monomer. Furthermore, we show that the minimal functional monomeric unit in the FH2 domain, termed the bridge element, can be inhibited by isolated monomeric DID. NMR data on the bridge-DID system revealed that at least one of the two actin-binding sites on the bridge element is accessible to actin monomer in the inhibited state.

Conclusions/Significance

Our findings suggest that autoinhibition in the native DRF dimer involves steric hindrance with the actin filament. Although the structure of a full-length DRF would be required for clarification of the presented models, our work here provides the first structural insights into the mechanism of the DRF autoinhibition.     

Crystal structure of a T cell receptor Valpha11 (AV11S5) domain: new canonical forms for the first and second complementarity determining regions.

We describe the X-ray crystallographic structure of a murine T cell receptor (TCR) Valpha domain ("Valpha85.33"; AV11S5-AJ17) to 1.85 A resolution. The Valpha85.33 domain is derived from a TCR that recognizes a type II collagen peptide associated with the murine major histocompatibility complex (MHC) class II molecule, I-A(q). Valpha85.33 packs as a Valpha-Valpha homodimer with a highly symmetric monomer-monomer interface. The first and second complementarity determining regions (CDR1 and CDR2) of this Valpha are shorter than the CDRs corresponding to the majority of other Valpha gene families, and three-dimensional structures of CDRs of these lengths have not been described previously. The CDR1 and CDR2 therefore represent new canonical forms that could serve as templates for AV11 family members. CDR3 of the Valpha85.33 domain is highly flexible and this is consistent with plasticity of this region of the TCR. The fourth hypervariable loop (HV4alpha) of AV11 and AV10 family members is one residue longer than that of other HV4alpha regions and shows a high degree of flexibility. The increase in length results in a distinct disposition of the conserved residue Lys68, which has been shown in other studies to play a role in antigen recognition. The X-ray structure of Valpha85.33 extends the database of canonical forms for CDR1 and CDR2, and has implications for antigen recognition by TCRs that contain related Valpha domains.     

Molecular mechanism for regulation of the human mitochondrial branched-chain alpha-ketoacid dehydrogenase complex by phosphorylation

The human mitochondrial branched-chain alpha-ketoacid dehydrogenase complex (BCKDC) is a 4 MDa macromolecular machine comprising three catalytic components (E1b, E2b, and E3), a kinase, and a phosphatase. The BCKDC overall activity is tightly regulated by phosphorylation in response to hormonal and dietary stimuli. We report that phosphorylation of Ser292-alpha in the E1b active site channel results in an order-to-disorder transition of the conserved phosphorylation loop carrying the phosphoryl serine. The conformational change is triggered by steric clashes of the phosphoryl group with invariant His291-alpha that serves as an indispensable anchor for the phosphorylation loop through bound thiamin diphosphate. Phosphorylation of Ser292-alpha does not severely impede the E1b-dependent decarboxylation of alpha-ketoacids. However, the disordered loop conformation prevents phosphorylated E1b from binding the E2b lipoyl-bearing domain, which effectively shuts off the E1b-catalyzed reductive acylation reaction and therefore completely inactivates BCKDC. This mechanism provides a paradigm for regulation of mitochondrial alpha-ketoacid dehydrogenase complexes by phosphorylation.     

Redox status of the eye lens: a regional study

The aim of this work was to study the regional variation of some antioxidant systems in calf lens. Specific lens regions of nearly same age were obtained by a microsectioning technique, and the concentration of reduced and oxidized glutathione, protein sulfhydryl groups, and iron were measured in each lens region. The concentration of reduced glutathione, the major redox buffer in lens, exponentially decreased from the cortical regions to the nucleus. In contrast, the concentration of protein sulfhydryl groups gradually increased from the cortex toward the nucleus. The protein-bound disulfides remained constant throughout the lens. Iron was concentrated in the outer cortical region. The results show that the most dynamic redox-active zone in the lens is the subcapsular cortical region where the oxidant flux meets a highly reducing environment containing a potent redox catalyst.     

Structural analysis of Xanthomonas XopD provides insights into substrate specificity of ubiquitin-like protein proteases

XopD (Xanthomonas outer protein D), a type III secreted effector from Xanthomonas campestris pv. vesicatoria, is a desumoylating enzyme with strict specificity for its plant small ubiquitin-like modifier (SUMO) substrates. Based on SUMO sequence alignments and peptidase assays with various plant, yeast, and mammalian SUMOs, we identified residues in SUMO that contribute to XopD/SUMO recognition. Further predictions regarding the enzyme/substrate specificity were made by solving the XopD crystal structure. By incorporating structural information with sequence alignments and enzyme assays, we were able to elucidate determinants of the rigid SUMO specificity exhibited by the Xanthomonas virulence factor XopD.     

Computational de novo design of a four-helix bundle protein—DND_4HB

The de novo design of proteins is a rigorous test of our understanding of the key determinants of protein structure. The helix bundle is an interesting de novo design model system due to the diverse topologies that can be generated from a few simple α-helices. Previously, noncomputational studies demonstrated that connecting amphipathic helices together with short loops can sometimes generate helix bundle proteins, regardless of the bundle''s exact sequence. However, using such methods, the precise positions of helices and side chains cannot be predetermined. Since protein function depends on exact positioning of residues, we examined if sequence design tools in the program Rosetta could be used to design a four-helix bundle with a predetermined structure. Helix position was specified using a folding procedure that constrained the design model to a defined topology, and iterative rounds of rotamer-based sequence design and backbone refinement were used to identify a low energy sequence for characterization. The designed protein, DND_4HB, unfolds cooperatively (T_m >90°C) and a NMR solution structure shows that it adopts the target helical bundle topology. Helices 2, 3, and 4 agree very closely with the design model (backbone RMSD = 1.11 Å) and >90% of the core side chain χ1 and χ2 angles are correctly predicted. Helix 1 lies in the target groove against the other helices, but is displaced 3 Å along the bundle axis. This result highlights the potential of computational design to create bundles with atomic-level precision, but also points at remaining challenges for achieving specific positioning between amphipathic helices.