Cyclic diguanylic acid behaves as a host molecule for planar intercalators

AbstractCyclic ribodiguanylic acid, c-(GpGp), is the endogenous effector regulator of cellulose synthase. Its three-dimensional structure from two different crystal forms (tetragonal and trigonal) has been determined by X-ray diffraction analysis at 1 Å resolution. In both crystal forms, two independent c-(GpGp) molecules associate with each other to form a self-intercalated dimer. A hydrated cobalt ion is found to coordinate to two N7 atoms of adjacent guanines, forcing these two guanines to destack with a large dihedral angle (32°), in the dimer of the tetragonal form. This metal coordination mechanism may be relevant to that of the anticancer drug cisplatin. Moreover, c-(GpGp) exhibits unusual spectral properties not seen in any other cyclic dinucleotide. It interacts with planar organic intercalator molecules in ways similar to double helical DNA. We propose a cage-like model consisting of a tetrameric c-(GpGp) aggregate in which a large cavity (‘host’) is generated to afford a binding site for certain planar intercalators (‘guests’)

Directed Evolution of a Lysosomal Enzyme with Enhanced Activity at Neutral pH by Mammalian Cell-Surface Display

SummaryHuman β-glucuronidase, due to low intrinsic immunogenicity in humans, is an attractive enzyme for tumor-specific prodrug activation, but its utility is hindered by low activity at physiological pH. Here we describe the development of a high-throughput screening procedure for enzymatic activity based on the stable retention of fluorescent reaction product in mammalian cells expressing properly folded glycoproteins on their surface. We utilized this procedure on error-prone PCR and saturation mutagenesis libraries to isolate β-glucuronidase tetramers that were up to 60-fold more active (kcat/Km) at pH 7.0 and were up to an order of magnitude more effective at catalyzing the conversion of two structurally disparate glucuronide prodrugs to anticancer agents. The screening procedure described here can facilitate investigation of eukaryotic enzymes requiring posttranslational modifications for biological activity

A Lon-Like Protease with No ATP-Powered Unfolding Activity

<div><p>Lon proteases are a family of ATP-dependent proteases involved in protein quality control, with a unique proteolytic domain and an AAA⁺ (ATPases associated with various cellular activities) module accommodated within a single polypeptide chain. They were classified into two types as either the ubiquitous soluble LonA or membrane-inserted archaeal LonB. In addition to the energy-dependent forms, a number of medically and ecologically important groups of bacteria encode a third type of Lon-like proteins in which the conserved proteolytic domain is fused to a large N-terminal fragment lacking canonical AAA⁺ motifs. Here we showed that these Lon-like proteases formed a clade distinct from LonA and LonB. Characterization of one such Lon-like protease from <em>Meiothermus taiwanensis</em> indicated that it formed a hexameric assembly with a hollow chamber similar to LonA/B. The enzyme was devoid of ATPase activity but retained an ability to bind symmetrically six nucleotides per hexamer; accordingly, structure-based alignment suggested possible existence of a non-functional AAA-like domain. The enzyme degraded unstructured or unfolded protein and peptide substrates, but not well-folded proteins, in ATP-independent manner. These results highlight a new type of Lon proteases that may be involved in breakdown of excessive damage or unfolded proteins during stress conditions without consumption of energy.</p> </div

Two LonC-specific insertions.

<p>(A) Sequence alignment of selected Lon proteases (LonA and LonB members are indicated accordingly) centered on the loop between helix α3 (see text), which houses the catalytic lysine (K625 in <i>M. taiwanensis</i>; close triangle), and strand β9 in the protease domain. Open triangles mark the insertion region. (B) Alignment detail of the C-terminal region of Lon protease domain focused on helix α5. (C) Sequence alignment of MtaLonC and TonLonB. Regions corresponding to the Walker A and B motifs of TonLonB are outlined in blue and red boxes, respectively. The two LonC-specific insertions is in black box. The MtaLonC residues corresponding to the sensor-1, sensor-2, and Arg finger of TonLonB are in boxes of yellow, purple, and green colors, respectively, for comparison (see text). The transmembrane regions of TonLon are underlined. Conserved residues are highlighted in black blocks. Catalytic dyad residues are marked with asterisks. (D) Locations of the two LonC-specific insertions, in the α3-β9 loop (colored in magenta) and helix α5 (pink) of the Lon protease domain (green), mapped onto a surface representation of hexameric TonLon (PDB code 3K1J).</p