Evidence for DNA-binding domain--ligand-binding domain communications in the androgen receptor

DNA binding as well as ligand binding by nuclear receptors has been studied extensively. Both binding functions are attributed to isolated domains of which the structure is known. The crystal structure of a complete receptor in complex with its ligand and DNA-response element, however, has been solved only for the peroxisome proliferator-activated receptor γ (PPARγ)-retinoid X receptor α (RXRα) heterodimer. This structure provided the first indication of direct interactions between the DNA-binding domain (DBD) and ligand-binding domain (LBD). In this study, we investigated whether there is a similar interface between the DNA- and ligand-binding domains for the androgen receptor (AR). Despite the structural differences between the AR- and PPARγ-LBD, a combination of in silico modeling and docking pointed out a putative interface between AR-DBD and AR-LBD. The surfaces were subjected to a point mutation analysis, which was inspired by known AR mutations described in androgen insensitivity syndromes and prostate cancer. Surprisingly, AR-LBD mutations D695N, R710A, F754S, and P766A induced a decrease in DNA binding but left ligand binding unaffected, while the DBD-residing mutations K590A, K592A, and E621A lowered the ligand-binding but not the DNA-binding affinity. We therefore propose that these residues are involved in allosteric communications between the AR-DBD and AR-LBD.     

Disruption of sscR encoding a γ-butyrolactone autoregulator receptor in Streptomyces scabies NBRC 12914 affects production of secondary metabolites

We report the cloning and sequence analysis of a gamma-butyrolactone autoregulator regulatory island that includes an sscR gene encoding the gamma-butyrolactone autoregulator receptor from Streptomyces scabies NBRC 12914, a plant pathogenic strain. gamma-Butyrolactone autoregulators trigger secondary metabolism, and sometimes morphological differentiation in the Gram-positive genus Streptomyces through binding to a specific autoregulator receptor. This gene cluster showed close similarity to other regulatory islands of Streptomyces origin that are responsible for the control of secondary metabolism. The recombinant SscR protein expressed in Escherichia coli prefers a gamma-butyrolactone autoregulator containing a long C-2 side chain and beta-hydroxyl group at the C-6 position. An inactivation experiment confirmed that this gamma-butyrolactone autoregulator receptor was involved in secondary metabolism but had no effects on the morphological differentiation. In the sscR-deleted mutant, the binding activity of the gamma-butyrolactone autoregulator was completely abolished, suggesting that its primary role is to detect the presence of an autoregulator in the environment. HPLC analysis of the culture broth showed that some peaks disappeared and new peaks that were not present in the broth of the wild-type strain appeared.     

Site-directed mutagenesis of the A-factor receptor protein:: Val-41 important for DNA-binding and Trp-119 important for ligand-binding

The A-factor receptor protein (ArpA) plays a key role in the regulation of secondary metabolism and cellular differentiation in Streptomyces griseus. ArpA binds the target DNA site forming a 22 bp palindrome in the absence of A-factor, and exogenous addition of A-factor to the ArpA–DNA complex immediately releases ArpA from the DNA. An amino acid (aa) replacement at Val-41 to Ala in an α-helix–turn–α-helix (HTH) motif at the N-terminal portion of ArpA abolished DNA-binding activity but not A-factor-binding activity, suggesting the involvement of this HTH in DNA-binding. On the other hand, an aa replacement at Trp-119 to Ala generated a mutant ArpA that was unable to bind A-factor, thus resulting in an A-factor-insensitive mutant that bound normally to its target DNA in both the presence and absence of A-factor. These data suggest that ArpA consisting of two functional domains, one for HTH-type DNA-binding at the N-terminal portion and one for A-factor-binding at the C-terminal portion, is a member of the LacI family. Consistent with this, two ArpA homologues, CprA and CprB, from Streptomyces coelicolor A3(2), each of which contains a very similar aa sequence of the HTH to that of ArpA, also recognized and bound the same DNA target. However, neither CprA nor CprB recognized A-factor, probably due to much less similarity in the C-terminal domains.     

Gamma-butyrolactones: Streptomyces signalling molecules regulating antibiotic production and differentiation

Small signalling molecules called gamma-butyrolactones are mainly produced by Streptomyces species in which they regulate antibiotic production and morphological differentiation. Their molecular mechanism of action has recently been unravelled in several streptomycetes, revealing a diverse and complex system. Gamma-butyrolactones and their receptors also occur in some other Actinobacteria, suggesting that this is a general regulatory system for antibiotic production. The gamma-butyrolactones bind to receptors, many of which are involved in regulation of specific antibiotic biosynthesis clusters. The importance of understanding how secondary metabolites are regulated and how environmental and physiological signals are sensed highlights the relevance of studying this system.     

Structural insight into the specific interaction between murine SHPS-1/SIRP alpha and its ligand CD47

SRC homology 2 domain-containing protein tyrosine phosphatase substrate 1 (SHPS-1 or SIRPα/BIT) is an immunoglobulin (Ig) superfamily transmembrane receptor and a member of the signal regulatory protein (SIRP) family involved in cell-cell interaction. SHPS-1 binds to its ligand CD47 to relay an inhibitory signal for cellular responses, whereas SIRPβ, an activating member of the same family, does not bind to CD47 despite sharing a highly homologous ligand-binding domain with SHPS-1. To address the molecular basis for specific CD47 recognition by SHPS-1, we present the crystal structure of the ligand-binding domain of murine SHPS-1 (mSHPS-1). Folding topology revealed that mSHPS-1 adopts an I2-set Ig fold, but its overall structure resembles IgV domains of antigen receptors, although it has an extended loop structure (C′E loop), which forms a dimer interface in the crystal. Site-directed mutagenesis studies of mSHPS-1 identified critical residues for CD47 binding including sites in the C′E loop and regions corresponding to complementarity-determining regions of antigen receptors. The structural and functional features of mSHPS-1 are consistent with the human SHPS-1 structure except that human SHPS-1 has an additional β-strand D. These results suggest that the variable complementarity-determining region-like loop structures in the binding surface of SHPS-1 are generally required for ligand recognition in a manner similar to that of antigen receptors, which may explain the diverse ligand-binding specificities of SIRP family receptors.