Another kind of dynamics: Ubiquitin noncovalently dimerizes with a dissociation constant of approximately 5 mM. The two subunits adopt an array of relative orientations, utilizing an interface also for binding to other proteins (see picture). Quaternary fluctuation among members of the dimer ensemble constitutes a different kind of dynamics that complements the tertiary dynamics of each ubiquitin subunit.

The flagellum and the injectisome enable bacterial locomotion and pathogenesis, respectively. These nanomachines assemble and function using a type III secretion system (T3SS). Exported proteins are delivered to the export apparatus by dedicated cytoplasmic chaperones for their transport through the membrane. The structural and mechanistic basis of this process is poorly understood. Here we report the structures of two ternary complexes among flagellar chaperones (FliT and FliS), protein substrates (the filament-capping FliD and flagellin FliC), and the export gate platform protein FlhA. The substrates do not interact directly with FlhA; however, they are required to induce a binding-competent conformation to the chaperone that exposes the recognition motif featuring a highly conserved sequence recognized by FlhA. The structural data reveal the recognition signal in a class of T3SS proteins and provide new insight into the assembly of key protein complexes at the export gate.

Ubiquitin is a small signaling protein in cells and is highly conserved throughout the eukaryotes. Ubiquitin interacts with myriad partner proteins that contain one or more ubiquitin-binding domains (UBDs). To achieve multivalent binding with several UBDs, ubiquitins are often covalently linked by an isopeptide bond between the C-terminal carboxyl group of one ubiquitin and a primary amine in another; [1a,b] the two subunits in a di-ubiquitin are referred to as the proximal unit and the distal unit, respectively. All seven lysines and the N-terminus of ubiquitin can participate in the isopeptide bond.[2] In tandem, multiple ubiquitins can be linked up to form a poly(ubiquitin).Depending on the site of the linkage, di-and poly(ubiquitin)s can display distinct quaternary structures, which may account for their linkage-specific functions. [1a,b] Among the linkages, Lys11, Lys48, and Lys63-linked poly(ubiquitin)s are best characterized: Lys63-linked poly(ubiquitin) is involved in cellular events such as endocytosis and DNA repair, while both Lys11-and Lys48-linked poly(ubiquitin)s can signal for proteosomal degradation. [1a,b] In crystal, Lys48-linked diubiquitin mostly adopts a closed conformation, burying hydrophobic residues around Ile44 in both subunits; [3a,b, 4] Lys63-linked di-ubiquitin adopts an open extended structure; [5a,b, 6] while Lys11-linked di-ubiquitin displays intermediate subunit separation. [7,8] Structural heterogeneity has also been observed for diand poly(ubiquitin)s with the same linkage. Lys48-linked diubiquitin has been crystallized in multiple forms, one of which actually adopts an open conformation ( Figure S1a), [4] while in solution at a neutral pH value, approxaimely15 % adopts the open conformation. [9] Although adopting an extended conformation in crystals, [5a,b] a highly compact structure was deduced for a sub-population of Lys63-linked di-ubiquitin from small-angle X-ray scattering data in solution.[6] For Lys11-linked di-ubiquitin, chains in a single asymmetric unit of crystal structure display substantial variations with root mean square (rms) variations over 6 ( Figure S1b). [8] In a quest to understand what affords the multitude of quaternary structures and to elucidate a linkage-structure relationship for poly(ubiquitin), we serendipitously discovered that free ubiquitin dimerizes noncovalently in solution. At increasing protein concentration, a subset of peaks of ubiquitin shift progressively (Figure 1 a and S2), corresponding to residues 8, 13, 44, 45, 46, 49, 67, 68, 70, 71, and 73, which are located at the b-sheet region of the protein and form a contiguous surface (Figure 1 b). Plotting the chemical shift values measured at 30 8C over protein concentrations, the

Variants within the human UGT1A1 gene are associated with irinotecan induced severely adverse reactions and hyperbilirubinemia. Intra-ethnic differences in the genetic variation and haplotypes of UGT1A1 gene have been analyzed in the present study. Relationship between the concentrations of total serum bilirubin (T-bil) and haplotype structure of UGT1A1 in healthy people were also evaluated. We genotyped five functional polymorphisms including À3279T4G and À3156G4A in the enhancer region, (TA)647 in the TATA box, and 211G4A (G71R), 686C4A (P229Q) in the exon1 region of UGT1A1 in three groups of healthy Chinese ethnic populations, consisting of 264 subjects of She origin, 539 of Han origin and 273 of Dong origin. The distribution of À3279T4G, (TA)647, 211G4A of UGT1A1 differed greatly as between the three ethnic groups. All of six haplotypes differed considerably between at least two of the three groups, which highlighted the need to analyze clinically irinotecan toxicity relevant SNPs and haplotypes in a variety of different racial groups within the Chinese population. Total bilirubin concentration in homozygous carriers of the À3279G and (TA)7 allele were significantly higher than those in heterozygous carriers or homozygous carriers of wild-type alleles. Carriers of the variant haplotypes (À3279G; À3156A; (TA)7; 211G; 686C) had higher serum T-Bil concentrations compared with the other groups. Our results indicate that heterogeneity among different ethnic populations is possibly the result of microevolution and is relevant to studies into the effect of tailored drug treatment.

Selective methyl labeling is an extremely powerful approach to study the structure, dynamics and function of biomolecule systems by NMR. Despite spectacular progress in the field, such studies are still rather limited in number. One of the main obstacles remains the assignment of the methyl resonances, which is labor intensive and error prone. Typically, NOESY crosspeak patterns are manually correlated to the available crystal structure or an in silico template model of the protein. Here, we propose Methyl Assignment by Graphing Inference Construct (MAGIC), an exhaustive search algorithm with no peak network definition requirement. In order to overcome the combinatorial problem, the exhaustive search is performed locally, i.e. for a small number of methyls connected through-space according to experimental 3D methyl NOESY data. The local network approach drastically reduces the search space. Only the best local assignments are combined together to provide the final output. Assignments that match the data with equivalent – or slightly lower - scores are made available to the user for cross-validation by additional experiments such as methyl-amide NOEs. Several NMR datasets for proteins in the 25–50 kDa range were used during development and for performance evaluation against the manually assigned data. We show that the algorithm is robust, reliable and greatly speeds up the methyl assignment task.

Proteins interact with each other to fulfill their functions. The importance of weak protein-protein interactions has been increasingly recognized. However, owing to technical difficulties, ultra-weak interactions remain to be characterized. Phosphorylation can take place via a K(D)≈25 mM interaction between two bacterial enzymes. Using paramagnetic NMR spectroscopy and with the introduction of a novel Gd(III)-based probe, we determined the structure of the resulting complex to atomic resolution. The structure accounts for the mechanism of phosphoryl transfer between the two enzymes and demonstrates the physical basis for their ultra-weak interaction. Further, molecular dynamics (MD) simulations suggest that the complex has a lifetime in the micro- to millisecond regimen. Hence such interaction is termed a fleeting interaction. From mathematical modeling, we propose that an ultra-weak fleeting interaction enables rapid flux of phosphoryl signal, providing a high effective protein concentration.

Intracellular nicotinamide phosphoribosyltransferase (iNAMPT) in neuron has been known as a protective factor against cerebral ischemia through its enzymatic activity, but the role of central extracellular NAMPT (eNAMPT) is not clear. Here we show that eNAMPT protein level was elevated in the ischemic rat brain after middle-cerebral-artery occlusion (MCAO) and reperfusion, which can be traced to at least in part from blood circulation. Administration of recombinant NAMPT protein exacerbated MCAO-induced neuronal injury in rat brain, while exacerbated oxygen-glucose-deprivation (OGD) induced neuronal injury only in neuron-glial mixed culture, but not in neuron culture. In the mixed culture, NAMPT protein promoted TNF-α release in a time- and concentration-dependent fashion, while TNF-α neutralizing antibody protected OGD-induced, NAMPT-enhanced neuronal injury. Importantly, H247A mutant of NAMPT with essentially no enzymatic activity exerted similar effects on ischemic neuronal injury and TNF-α release as the wild type protein. Thus, eNAMPT is an injurious and inflammatory factor in cerebral ischemia and aggravates ischemic neuronal injury by triggering TNF-α release from glia cells, via a mechanism not related to NAMPT enzymatic activity.

Many steroids are important pharmaceutically active compounds, while cytochrome P450 monooxygenases (CYPs) are attractive enzymes for applications in steroidal drug synthesis. However, the catalytic efficiency of existing P450s is not routinely high enough, as well as the molecular basis for selectivity control is unclear, which severely restrict their real applications. Here, a 16β steroid-hydroxylase CYP109B4 from Bacillus sonorensis is identified with excellent selectivity and activity. The crystallization and structural analysis of CYP109B4 reveal potential three "hotspot" residues (V84, V292, and S387) responsible for selectivity control. Then, guided by the sequence−function relationships revealed from the mutability landscape construction on the three residues, focused rational iterative site-specific mutagenesis (FRISM) and limited iterative saturation mutagenesis were performed, which provide variant B4-M7 (L240V/S387F/V84L/V292S/I291T/M290F/F294I) with completely switched regioselectivity from 16β to 15β. The subsequent computational analysis uncovers insights into the substrate binding modes in CYP109B4 and its variants, which further confirms the critical role of the "hotspot" residues for selectivity control. Finally, the generality of conserved-"hotspots"mediated selectivity control is demonstrated by performing scaffold sampling between a panel of CYP109B members. Overall, in addition to the present chemical results, our study provides guidance in rationally designing more excellent P450 biocatalysts for potential practical (industrial) applications.