The histidine-rich proteins in prokaryotes and their biological significance

Abstract

Special stretches of sequence with low complexity, highly rich in one certain residue, such as glutamine, asparagines, glutamic acid and histidine, to fulfill certain unique functions, are defined as single-residue-rich sequence (SRRs). Increasing SRRs containing proteins have recently been characterized and some of them have been indentified to be associated with immune system diseases or neuro-degenerative. A systematic and comprehensive analysis on the relationship between the occurrence of histidine-rich motifs (HRMs) and the functions of corresponding proteins have been overlooked.

In this thesis, proteome sequences of 675 prokaryotes including 50 archeae proteome sequences and 625 bacteria were examined and analyzed for HRMs. The HRMs are shown to be extensively distributed in prokaryotic proteomes and the majority (62%) of them is identified to be involved in metal homeostasis. Intriguingly, HRMs are essentially absent from obligate intracellular pathogenic species such as Rickettsiales, Chlamydiae and Tenericutes but are frequently found in the proteomes of Rhizobiales and Burkholderiales, both of which habitat in soils, indicative of environmental habitat-related occurrence of HRMs. Based on the primary sequence to explore the histidine-rich proteins, the present approach could be extended to apply for searching other single-residue-rich proteins, which may shed lights on gaining a further understanding about relationship between the proteins’ sequences and their functions.

A novel group of globally histidine-rich proteins was discovered, among which a histidine-rich protein, bacterioferritin-associated ferredoxin ((BFD)-like [2Fe-2S]) protein from Rhodopseudomonas Palustris BisB18 (termed as BFD shortly) was digged out. The BFD protein consists of a Fe-S cluster domain (FeSD) at the N-terminus and an extremely histidine-rich domain (HRD) at the C-terminus. The intact protein BFD as well as its histidine-rich domain (HRD) was over-expressed, purified and characterized and the effects of metal binding on BFD and HRD were examined in this work.

The intact protein BFD presents as a 20 mer whereas the HRD protein exists as a monomer in solution. However, the CD spectrum of BFD showed the presence of both α-helix and β-sheet in the structure of BFD. The CD spectrum of HRD demonstrated that an extremely large portion of the structure of HRD was random coils, which indicated that the most of the α-helix and β-sheet predominately were located in the Fe-S cluster domain (FeSD) of BFD. It also indicated that HRD adopted a very flexible conformation, which was in good agreement with the results that obtained from the 2D 1H-15N HSQC spectrum of HRD. Isothermal titration calorimetry and equilibrium dialysis revealed that HRD possessed a large binding capacity to divalent metal ions (up to 9 Ni2+, 5 Zn2+ and 4 Co2+ respectively). The E. coli cells over-expressed with the HRD protein showed a significantly evaluated metal resistance to the toxic Ni and Co ions. The amounts of meals in these cells were determined to be approximate 3-5 fold higher than those in the control groups. These results of HRD taken together suggest the characteristics of common globally histidine-rich proteins.