Structure And Function Of The Novel Transcriptional Factor MrkH

Klebsiella pneumonia is an opportunistic pathogen of nosocomial infections.Its multiple antibiotic resistanceis largely a result of the bacteria’s ability to form biofilms on biomedical devices. Biofilm formation of bacterial is regulated by a variety of factors. An important physiological change that occurs within bacterial cells during the conversion from planktonic to biofilm life-styles is the enhancement of levels of the second messenger 3’,5’-cyclic two guanine nucleotides (c-di-GMP). Key to K. pneumonia biofilm development are mannose-resistant klebsiella-like (Mrk) hemagglutinins or "Mrk proteins". These proteins are encoded by the mrkABCDF operon and form type 3 fimbriae, which has been demonstrated that play an important role in biofilm formation of K. pneumoniae. Fimbriae (or pill) also help bacterial pathogens adhere to host cells for successful infection. Type 1 and type 3 fimbriae are well characterized in K. pneumoniae, the MrkA protein is the major fimbrial subunit which is polymerized to form the helical frnbrial shaft, MrkB and MrkC function as the periplasmic chaperone and translocate MrkA and MrkD to the adhesion filaments respectively, MrkD forms the tip adhesion subunit. Type 3 fimbriae can adhere to extracellular matrix proteins such as type V collagen, as well as the basement membrane regions of pulmonary epithelia.Recent studies show that the expression of type 3 pili which are encoded by the mrk gene cluster is regulated by a c-di-GMP level-dependent transcription factor-MrkH. c-di-GMP is able to directly modulate the activities of MrkH which is a novel transcription factor that control the expression of genes involved in biofilm formation. MrkH consists of two domains:an N terminal YcgR-N like domain and a C terminal PilZ domain. PilZ domain is a typical c-di-GMP binding motif, while the function of YcgR-N like domain remains obscure.To our knowledge, MrkH is the first PilZ-containing protein to function in DNA binding, while there are a lot of controversy about how MrkH recognize the DNA sequence.Wilksch et.al first discovered MrkH is a novel c-di-GMP-dependent transcriptional activator in 2011, they continued further research and located the DNA-binding sequence in the regulatory promoter region of mrkHI and mrkA. While Steven Cleg et.al found that MrkH and MrkI together can regulate the expression of type 3 fimbriae more efficiently. MrkI is a classical DNA binding protein, mrkl locates next to mrkH and these two genes are co-transcribed. It is not clear whether MrkI is directly involved in the transcriptional regulation of MrkH until now. In this paper, we want to obtain the crystal structures of Apo-MrkH, MrkH/c-di-GMP and MrkH/DNA and investigate the DNA binding model of MrkH. We also comprehensively use of various experimental methods such as biochemistry, molecular biology, microbiology to reveal the molecular mechanisms of the regulation process of the type 3 fimbriae’s expression that is regulated by MrkH alone or MrkH together with MrkI.We obtained the crystal structure of MrkH/c-di-GMP at 2.3 Angstrom and compared the MrkH/c-di-GMP complex structure with other homologous structures. The structures are very similar and the most difference is in the PilZ domain. MrkH protein exists mainly in monomeric form, and its PilZ domain contains many basic residues regions which are likely to combine a number of negatively charged molecules, such as DNA molecules. We carefully analyzethe crystal structure of MrkH/c-di-GMP and find the ineraction mode of MrkH and c-di-GMP, we also use ITC to verify the specific contribution of conservative residues of MrkH for its c-di-GMP binding.We use EMSA to locate which motif of MrkH contributes the DNA binding, and find that the PilZ domain of MrkH can bind to the DNA alone. It has a unique DNA-binding mode: one longer DNA sequence can combine with PilZ domain molecules continuously. It infers that MrkH_PilZ binds to DNA in a nonspecific way. We incubate the MrkH protein and the 12bp specific MrkH ’box’ for crystal screen, finally we obtain a new structure of MrkH/c-di-GMP complex. Compare with the frist structure, we find the c-di-GMP binding loop of the second structure will be more curvedin the presence of DNA, which makes the YcgR-N and PilZ domains closer to each other. In order to locate the DNA binding region of MrkH, the vacuum electrostatics of MrkH/c-di-GMP structure is carefully analyzed. We mutated the basic residues of the potential binding region for further investigation by site-directed mutagenesis method. EMSA experiments show that the mutant MrkH proteins are lost or diminished DNA binding activity. At the same time we also use FP method to calculate the Kd values of various MrkH mutant binding to DNA. These results are consistent and show that MrkH binds to non-specific DNA sequence primarily through its positive charge region The DNA binding-related key residues mainly locate in the PilZ domain of MrkH. We also try to use the latest technology Cryo-EM to get the structure of MrkH and DNA complex. After many efforts, we could not get good quality of single particles, which also verified the MrkH/DNA complex is very inhomogeneous.MrkH is co-transcribed with a HTH protein-MrkI, we assume that MrkHI complex will regulate the expression of type 3 fimbriae in K. pneumoniae. But the expression of MrkI always failed, so we use MBP fusion tag to get soluble MrkI. It seems that MrkI and MrkH can form a complex, and MrkH mainly through its N-terminal domain to interact with MrkI from our in vitro pull down experiments. MrkI can also binds to mrkABCDF gene cluster and its own promoter regulatory regions. As a typical DNA binding protein, it suggests that MrkI binds to DNA in a specific way.According to the above results, we propose the molecular mechanisms of MrkHI self-regulation and the regulation of type 3 fimbriae’s expression in K. pneumoniae:one model is MrkH and MrkI form complex first, the MrkHI complex recognizes the promoter region of mrkABCDF and mrkHI. MrkI can recognize the specific DNA sequence simultaneously. C-di-GMP binding MrkH resulted in a relative position of the two terminal domains of MrkH and makes the MrkHI complex conformational changes, which makes the transcriptional regulation more efficiently at last. Another model requires anot-yet- identified protein X that canspecifically recognize ’MrkH box’. Once protein X binds to ’MrkH box’, it will recruit MrkH and MrkI to form an active ternary complex which finally activates transcription of mrkABCDF and mrkHI. c-di-GMP can binds to MrkH and makes the ternary complex conformational changes to affect the DNA binding of MrkHI and X. MrkH_PilZ domain is a very small structure with only 132 residues, yet acts as both a c-di-GMP receptor and a DNA binding motif It suggests that there are many undiscovered functions of PilZ domains. MrkI is a typical LuxR-transcription factor which contains a helix-turn-helix DNA binding domain, but its purification is unsuccessful. We first demonstrate that MrkH_ PilZ domain is a novel DNA-binding domain. However, the accurate DNA-binding model is still unclear. We can prove PilZ domains recognize nonspecific DNA sequence in vitro, but whether the intracellular c-di-GMP level affect the DNA-binding ability of MrkHI? No bacterial physiology data were conducted to shed lights on molecular mechanism of the gene transcription activation of MrkH. Therefore, we need more in vivo data to refine our novel findings.