Nucleotide binding by the widespread high-affinity cyclic di-GMP receptor MshEN domain.

C-di-GMP is a bacterial second messenger regulating various cellular functions. Many bacteria contain c-di-GMP-metabolizing enzymes but lack known c-di-GMP receptors. Recently, two MshE-type ATPases associated with bacterial type II secretion system and type IV pilus formation were shown to specifically bind c-di-GMP. Here we report crystal structure of the MshE N-terminal domain (MshEN1-145) from Vibrio cholerae in complex with c-di-GMP at a 1.37 Å resolution. This structure reveals a unique c-di-GMP-binding mode, featuring a tandem array of two highly conserved binding motifs, each comprising a 24-residue sequence RLGxx(L/V/I)(L/V/I)xxG(L/V/I)(L/V/I)xxxxLxxxLxxQ that binds half of the c-di-GMP molecule, primarily through hydrophobic interactions. Mutating these highly conserved residues markedly reduces c-di-GMP binding and biofilm formation by V. cholerae. This c-di-GMP-binding motif is present in diverse bacterial proteins exhibiting binding affinities ranging from 0.5 µM to as low as 14 nM. The MshEN domain contains the longest nucleotide-binding motif reported to date.

Structural Insights into the Distinct Binding Mode of Cyclic Di-AMP with SaCpaA_RCK.

Cyclic di-AMP (c-di-AMP) is a relatively new member of the family of bacterial cyclic dinucleotide second messengers. It has attracted significant attention in recent years because of the abundant roles it plays in a variety of Gram-positive bacteria. The structural features that allow diverse bacterial proteins to bind c-di-AMP are not fully understood. Here we report the biophysical and structural studies of c-di-AMP in complex with a bacterial cation-proton antiporter (CpaA) RCK (regulator of the conductance of K(+)) protein from Staphylococcus aureus (Sa). The crystal structure of the SaCpaA_RCK C-terminal domain (CTD) in complex with c-di-AMP was determined to a resolution of 1.81 Å. This structure revealed two well-liganded water molecules, each interacting with one of the adenine bases by a unique H2Olp-π interaction to stabilize the complex. Sequence blasting using the SaCpaA_RCK primary sequence against the bacterial genome database returned many CpaA analogues, and alignment of these sequences revealed that the active site residues are all well-conserved, indicating a universal c-di-AMP binding mode for CpaA_RCK. A proteoliposome activity assay using the full-length SaCpaA membrane protein indicated that c-di-AMP binding alters its antiporter activity by approximately 40%. A comparison of this structure to all other reported c-di-AMP-receptor complex structures revealed that c-di-AMP binds to receptors in either a "U-shape" or "V-shape" mode. The two adenine rings are stabilized in the inner interaction zone by a variety of CH-π, cation-π, backbone-π, or H2Olp-π interaction, but more commonly in the outer interaction zone by hydrophobic CH-π or π-π interaction. The structures determined to date provide an understanding of the mechanisms by which a single c-di-AMP can interact with a variety of receptor proteins, and how c-di-AMP binds receptor proteins in a special way different from that of c-di-GMP.

Backbone resonance assignments of the 54 kDa dimeric C-terminal domain of murine STING in complex with DMXAA.

The mammalian ER protein STING (stimulators of interferon genes) is an important innate immunity protein for linking detection of novel secondary messengers c-di-GMP, c-di-AMP, cGAMP or cytosolic dsDNA to the activation of TANK kinase 1 and its downstream interferon regulator factor 3. Recently quite a few of crystal structures representing different states of the C-terminal domain (CTD) of human and murine STING (hSTING and mSTING) in complex with c-di-GMP, cGAMP or DMXAA have been reported. However, the C-terminal 42 residues of STING-CTD, which may be important in mediating the downstream reactions, is invisible or absent in all reported X-ray structures. In addition, X-ray crystal structures may be subject to crystal packing force. Hence an alternate method of determining the structure and function of STING in a near physiological condition is essential. We now report the near complete backbone resonance assignments of the 54 kDa dimeric mSTING-CTD in complex with DMXAA, which is the first step in determining its complex structure and understanding why DMXAA, which is a very efficient agent for curing mouse cancer, is totally ineffective in human.

Structure of Stenotrophomonas maltophilia FeoA complexed with zinc: a unique prokaryotic SH3-domain protein that possibly acts as a bacterial ferrous iron-transport activating factor.

Iron is vital to the majority of prokaryotes, with ferrous iron believed to be the preferred form for iron uptake owing to its much better solubility. The major route for bacterial ferrous iron uptake is found to be via an Feo (ferrous iron-transport) system comprising the three proteins FeoA, FeoB and FeoC. Although the structure and function of FeoB have received much attention recently, the roles played by FeoA and FeoC have been little investigated to date. Here, the tertiary structure of FeoA from Stenotrophomonas maltophilia (Sm), a vital opportunistic pathogen in immunodepressed hosts, is reported. The crystal structure of SmFeoA has been determined to a resolution of 1.7 A using an Se single-wavelength anomalous dispersion (Se-SAD) approach. Although SmFeoA bears low sequence identity to eukaryotic proteins, its structure is found to adopt a eukaryotic SH3-domain-like fold. It also bears weak similarity to the C-terminal SH3 domain of bacterial DtxR (diphtheria toxin regulator), with some unique characteristics. Intriguingly, SmFeoA is found to adopt a unique dimer cross-linked by two zinc ions and six anions (chloride ions). Since FeoB has been found to contain a G-protein-like domain with low GTPase activity, FeoA may interact with FeoB through the SH3-G-protein domain interaction to act as a ferrous iron-transport activating factor.

Insights into the alkyl peroxide reduction pathway of Xanthomonas campestris bacterioferritin comigratory protein from the trapped intermediate-ligand complex structures.

Considerable insights into the oxidoreduction activity of the Xanthomonas campestris bacterioferritin comigratory protein (XcBCP) have been obtained from trapped intermediate/ligand complex structures determined by X-ray crystallography. Multiple sequence alignment and enzyme assay indicate that XcBCP belongs to a subfamily of atypical 2-Cys peroxiredoxins (Prxs), containing a strictly conserved peroxidatic cysteine (C(P)48) and an unconserved resolving cysteine (C(R)84). Crystals at different states, i.e. Free_SH state, Intra_SS state, and Inter_SS state, were obtained by screening the XcBCP proteins from a double C48S/C84S mutant, a wild type, and a C48A mutant, respectively. A formate or an alkyl analog with two water molecules that mimic an alkyl peroxide substrate was found close to the active site of the Free_SH or Inter_SS state, respectively. Their global structures were found to contain a novel substrate-binding pocket capable of accommodating an alkyl chain of no less than 16 carbons. In addition, in the Intra_SS or Inter_SS state, substantial local unfolding or complete unfolding of the C(R)-helix was detected, with the C(P)-helix remaining essentially unchanged. This is in contrast to the earlier observation that the C(P)-helix exhibits local unfolding during disulfide bond formation in typical 2-Cys Prxs. These rich experimental data have enabled us to propose a pathway by which XcBCP carries out its oxidoreduction activity through the alternate opening and closing of the substrate entry channel and the disulfide-bond pocket.

The nature of actinomycin D binding to d(AACCAXYG) sequence motifs.

Earlier studies by others had indicated that actinomycin D (ACTD) binds well to d(AACCATAG) and the end sequence TAG-3' is essential for its strong binding. In an effort to verify these assertions and to uncover other possible strong ACTD binding sequences as well as to elucidate the nature of their binding, systematic studies have been carried out with oligomers of d(AACCAXYG) sequence motifs, where X and Y can be any DNA base. The results indicate that in addition to TAG-3', oligomers ending with XAG-3' and XCG-3' all provide binding constants > or =1 x 10(7) M(-1) and even sequences ending with XTG-3' and XGG-3' exhibit binding affinities in the range 1-8 x 10(6) M(-1). The nature of the strong ACTD affinity of the sequences d(A1A2C3C4A5X6Y7G8) was delineated via comparative binding studies of d(AACCAAAG), d(AGCCAAAG) and their base substituted derivatives. Two binding modes are proposed to coexist, with the major component consisting of the 3'-terminus G base folding back to base pair with C4 and the ACTD inserting at A2C3C4 by looping out the C3 while both faces of the chromophore are stacked by A and G bases, respectively. The minor mode is for the G to base pair with C3 and to have the same A/chromophore/G stacking but without a looped out base. These assertions are supported by induced circular dichroic and fluorescence spectral measurements.

Binding of actinomycin D to single-stranded DNA of sequence motifs d(TGTCT(n)G) and d(TGT(n)GTCT).

Our recent binding studies with oligomers derived from base replacements on d(CGTCGTCG) had led to the finding that actinomycin D (ACTD) binds strongly to d(TGTCATTG) of apparent single-stranded conformation without GpC sequence. A fold-back binding model was speculated in which the planar phenoxazone inserts at the GTC site with a loop-out T base whereas the G base at the 3'-terminus folds back to form a basepair with the internal C and stacks on the opposite face of the chromophore. To provide a more concrete support for such a model, ACTD equilibrium binding studies were carried out and the results are reported herein on oligomers of sequence motifs d(TGTCT(n)G) and d(TGT(n)GTC). These oligomers are not expected to form dimeric duplexes and contain no canonical GpC sequences. It was found that ACTD binds strongly to d(TGTCTTTTG), d(TGTTTTGTC), and d(TGTTTTTGTC), all exhibiting 1:1 drug/strand binding stoichiometry. The fold-back binding model with displaced T base is further supported by the finding that appending TC and TCA at the 3'-terminus of d(TGTCTTTTG) results in oligomers that exhibit enhanced ACTD affinities, consequence of the added basepairing to facilitate the hairpin formation of d(TGTCTTTTGTC) and d(TGTCTTTTGTCA) in stabilizing the GTC/GTC binding site for juxtaposing the two G bases for easy stacking on both faces of the phenoxazone chromophore. Further support comes from the observation of considerable reduction in ACTD affinity when GTC is replaced by GTTC in an oligomer, in line with the reasoning that displacing two T bases to form a bulge for ACTD binding is more difficult than displacing a single base. Based on the elucidated binding principle of phenoxazone ring requiring its opposite faces to be stacked by the 3'-sides of two G bases for tight ACTD binding, several oligonucleotide sequences have been designed and found to bind well.

Looped out and perpendicular: deformation of Watson-Crick base pair associated with actinomycin D binding.

Many anticancer drugs interact directly with DNA to exert their biological functions. To date, all noncovalent, intercalating drugs interact with DNA exclusively by inserting their chromophores into base steps to form elongated and unwound duplex structures without disrupting the flanking base pairs. By using actinomycin D (ActD)-5'-GXC/CYG-5' complexes as examples, we have found a rather unusual interaction mode for the intercalated drug; the central Watson-Crick X/Y base pairs are looped out and displaced by the ActD chromophore. The looped-out bases are not disordered but interact perpendicularly with the base/chromophore and form specific H bonds with DNA. Such a complex structure provides intriguing insights into how ligand interacts with DNA and enlarges the repertoires for sequence-specific DNA recognition.