Unlike to ion pump bacteriorhodopsin homologues, microbial sensory rhodopsin-transducer molecular complexes served a key paradigm for exploring the chemistry of protein-protein interaction in signaling cascade. In 2003, freshwater cyanobacterium Anabaena (Nostoc) sp. PCC 7120 revealed a chromosomal gene encoding a sensory rhodopsin analogue. In contrast to haloarchaeal sensory rhodopsin, it does not contain any membrane transducer gene product. However, an adjacent gene product is indicated to be a soluble transducer molecule for this photoreceptor. The detailed molecular mechanism of photoactivated receptor mediated signaling is not known. The proposed model is not related to any known microbial sensory based phototaxis. However, it is analogous to visual photoreceptor, rhodopsin and to other G- protein coupled signaling via cytoplasmic components. Thus the role of this soluble putative transducer protein may establish a novel mode of signaling as shared by haloarchaea and eukarya. Interestingly, this putative soluble transducer molecule is present in a series of microbial species that do not contain sensory rhodopisn photoreceptor. Sequential and structural fold homology reveals such molecule without any established functional feature. Genome database search revealed their presence in broad microbial population including various pathogens. The homologue sequence of a transducer present in other sequences is referred to as domain of unidentified function [DUF], family. This family includes pathogens, such as Tropheryma, Actinomyces and Thermobifida. The atomic resolution structures of both photoreceptor and putative transducer are available. Structural motif based bioinformatics analysis has revealed that the transducer homologue may be classified as a member of a super family of microbial small carbohydrate binding domain. It is likely that it may serve as a novel carbohydrate binding module in a unique beta sandwich framework. Our preliminary data, based on unipolar localization suggests strong correlation and affinity to bacterial cellulose synthesis proteins that specifically localizes at pole. Recent NMR study has shown a eukaryotic like interaction of this transducer with DNA. Further, sequence- based analysis prediction along with initial data from PI's work supports the phosphor-accepting property of the transducer protein. Additionally, the phosphorylation leads to impair the unusual stability of transducer, suggesting its putative role in signaling. The unusual stable assembly and its segregation of this putative transducer protein would elucidate the signaling state of this DUF protein molecule. This project is aimed to relate the functional state of transducer as a phosphor-acceptor module and presence of a sugar binding motif and characterize the modulation of such a feature on receptor binding. The characterization of transducer's signaling state would broaden our understanding towards other DUF members including various pathogens as well.

Public Health Relevance

Integral membrane receptor activation and downstream signaling via protein-protein interaction are instrumental to understanding complex signaling pathways. The fresh water cyanobacterium, Anabaena (Nostoc) PCC 7120, encodes a sensory rhodopsin homologue. It is hypothesized that the photoreceptor interacts with a downstream gene product, a soluble transducer protein present in the same operon. The receptor mediated a signal transduction and a functional state of putative transducer is yet to be established. In ths project, we will examine the functional features (i.e., the carbohydrate (s) binding feature, phospho-transfer, lipid interaction, etc.) of a putative anabaena sensory transducer and investigate its modulation to binding to an anabaena sensory receptor. It will lead to identification and functional characterization of 'DUF' features of anabaena sensory receptor transducer homologues in a variety of microbes including pathogens, such as Tropheryma, Actinomyces and Thermobifida. Public heath relevance statement: Sensory microbial rhodopsins, in general, convey a signal via photoactivation of a membrane receptor followed by transduction cascade events via the protein-protein interaction. This work will establish that the putative transducer and its orthologs termed as a domain of an unidentified function (member of DUF1362 family) can serve as a carbohydrate binding module and can be phosphorylated by a protein kinase. This putative state can be linked to a functional state of the transducer protein. The DUF domain is present in several other microbes including pathogens. Understanding of such functional state(s) at the molecular level would be of high significance in enhancing our understanding of the cellular process inside living cells. Success of this study promises a depth of understanding of a DUF-categorized putative transducer protein and its interaction with a sensory receptor in cellular signaling. Such interactions drive fundamental membrane processes that are crucial to normal cellular function; as such, membrane dysfunctions are involved in myriad disease states.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Continuance Award (SC3)
Project #
5SC3GM113803-03
Application #
9206509
Study Section
Special Emphasis Panel (ZGM1-TWD-6 (SC))
Program Officer
Krasnewich, Donna M
Project Start
2015-05-01
Project End
2019-01-31
Budget Start
2017-02-01
Budget End
2018-01-31
Support Year
3
Fiscal Year
2017
Total Cost
$85,275
Indirect Cost
$17,775
Name
Bethune-Cookman College
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
078315918
City
Daytona Beach
State
FL
Country
United States
Zip Code
32114