A structural insight into the prokaryotic heat shock transcription regulatory protein σ32: an implication of σ32-DnaK interaction

The heat shock response mechanism is a very vital biochemical process and is mainly controlled by σ32 protein. The function of σ32 is temperature dependent and at lower temperatures σ32 is inactivated by its interactions with DnaK. This interaction is completely abolished above 42°C till date no molecular details of the interactions are available. In the present scenario, an attempt has been made to analyze first the predicted structure of σ32 obtained by comparative modeling techniques and then to study the interactions between σ32 and DnaK. From this molecular modeling study we could specifically identify the binding sites of the interactions of σ32 with DnaK which will enlighten the mechanism of regulation of its activity and stability by DnaK. Our study provides the idea for future mutational experiments in order to find out the possible roles of the amino acids of region2 and region3 of σ32 in stability as well as in binding with DnaK.


Background:
The proper conformation of proteins and hence cellular survival is challenged by stress conditions like extreme heat which results in a massive aggregation of proteins inside both eukaryotic and prokaryotic cells. This response called the 'heat shock response' leads to the induction of almost all the universally conserved 'heat shock genes' which encode chaperones, proteases and other stress related proteins. In E. coli, this regulation is mediated by rpoH gene product, the alternative sigma factor σ 32 . Its intracellular level is low and increases transiently after temperature up-shift [1]. The cellular concentration of σ 32 is tightly controlled at four different levels: transcription and translation of the rpoH and activity and stability of σ 32 protein. Heat induction of σ 32 mainly occurs at the post transcriptional level. An extended secondary structure in the rpoH transcript blocks translation at low temperatures [2,3]. Thermal melting of that structure permits ribosome entry followed by translation initiation. Once produced the fate of σ 32 is determined by its interaction with a number of other proteins including chaperones as DnaK, DnaJ, GrpE and GroEL/ES and proteases as ClpP family, HslUV, Lon and FtsH [4,5].
Under non-stress conditions, σ 32 is neutralized by an interaction with DnaK and DnaJ proteins. This interaction serves two regulatory functions. It inactivates σ 32 by preventing it from interaction with the RNA polymerase core enzyme and renders it susceptible to FtsH-mediated degradation [5, 6] (half-life of σ 32 is < 1min) as over expression of σ 32 is toxic [7]. Accumulation of unfolded proteins upon heat stress conditions titrates away the DnaK system, leaving behind free σ 32 , which associates with RNA polymerase and in turn initiates transcription of heat shock genes. Accumulation of σ 32 only occurs in the initial phase (induction phase) of the heat shock response where the levels and half-life of σ 32 increase transiently [1,8]. Elevated temperatures introduce a conformational change in σ 32 which specifically abolishes interaction with DnaK [9]. It is assumed that the amino acid residues 190-205 in σ 32 are responsible for σ 32 -specific function and become disordered at higher temperatures [9]. The structurally altered sigma factor is rapidly turned over by cellular proteases. As a consequence, the cellular level of σ 32 decreases and the heat shock response is shut off. Several experiments for studying the dynamical properties as fluorescence resonance energy transfer, fluorescence anisotropy measurements and hydrogen / deuterium exchange have suggested significant structural flexibility in the core of σ 32 [10]. This property is probably the main hindrance for the formation of good crystals and hence high resolution crystallographic 3D structure of the protein is still unavailable. As a result many structural and functional properties of σ 32 , its interactions with the chaperones as well as with the proteases are still obscure. In the present scenario an attempt has been made to analyze the structural biochemistry of σ 32 protein along with its interactions with DnaK. We report a three dimensional model of σ 32 built by homology modeling. We have docked the 3D structure of DnaK with the homology model of σ 32 . This docked model has been used to illuminate structural insight of the mechanism by which DnaK interacts with σ 32 and regulate its activity and stability.

Methodology:
The amino acid sequence of σ 32 protein from E. coli K-12 was collected from Uniprot (accession numbers P0AGB3). The amino acid sequence of the σ 32 was used to search Brookhaven Protein Data Bank (PDB) [11] for suitable template(s) to build homology model using the BLAST software tool [12]. The BLAST search picked up the crystal structure of T. thermophilus RNA polymerase holoenzyme (PDB Code: 2A6H F chain) as the template with 40% sequence identity with σ 32 from E. coli K-12. Modeler program in the Discovery Studio 2.5 Platform of the Accelrys was used to model the three dimensional structure of the σ 32 using the above template. The model of σ 32 protein was then subjected to energy minimization using CHARMM force fields [13] using steepest descent (SD) algorithm. The stereochemical qualities of the three dimensional models were then checked using PROCHECK [14], and ERRAT [15] which predicts it as a model of good quality and no residues were found to be present in the disallowed regions of the Ramachandran Plot [16]. In order to build σ 32 -DnaK complex, the modeled structure of σ 32 was docked with the crystal structure of DnaK protein (PDB Code: 1DKX) using the program GRAMM [17]. GRAMM produced seven different models of the σ 32 -DnaK protein complex. Among them the best structure of the complex was chosen on the basis of the biological relevance as present in literature [10]. The model of σ 32 -DnaK protein complex was then protonated at pH 7.5 using Accelrys Discovery Studio 2.5 and then subjected to 2000 cycles of energy minimization using CHARMM force fields with steepest descent (SD) algorithm until the structure of the σ 32 -DnaK protein complex reached the final energy derivative of 0.001 kcal / mole. The stereo-chemical qualities of the docked protein complex were again checked using PROCHECK and ERRAT and results were same as before. The system was solvated with water molecules. Then molecular dynamics (MD) simulations were performed on the docked structure to predict the favorable binding interactions between σ 32 and DnaK.
Initially the dynamics run was kept constant at 303K (30 • C). The σ 32 -DnaK complex was then heated until it reached the temperature 316K (43 • C). The same process has been followed for the modeled σ 32 protein (i.e., before docking to DnaK protein) at 303K and 316K temperatures. The final structures of σ 32 proteins as well as those of σ 32 -DnaK complex at 303K and after heating at 316K were then analyzed to find out the possible modes of binding. The two structures were superimposed using 'align and superimpose' module of Discovery Studio 2.5 platform to find out the structural changes of σ 32 due to temperature up shift.

Structure of sigma32
The σ 32 protein from E.coli is an all alpha protein with 284 amino acid residues. The protein has a total of thirteen helices (Figure 1 A). Structurally the protein can be considered to have four domains. All the domains are made up of helices connected together by loop regions. Overall the protein has similar structural arrangements of its secondary structural elements as in the protein RNA polymerase holoenzyme from T. thermophilus (PDB ID: 2A6H_F chain).

Structural changes of sigma32 with temperature
It has been a well established fact that in E. coli upon temperature increase from normal to heat shock temperature, cellular level of σ 32 protein increases [8]. Till date no detailed analysis of the conformational changes of σ 32 at elevated temperature has been elucidated. In order to account for the loss of σ 32 specific function from a structural perspective, the model of the σ 32 protein was stepwise heated from 32˚C to beyond 32˚C. Initially no structural changes were observed but at around 43˚C the spatial arrangement of the protein were found to be significantly altered as measured by the r.m.s.d values (10.696 Å) of the backbone atoms of the proteins at these two different temperatures. This clearly indicates a huge change in the conformation of the σ 32 protein at elevated temperature.

Interactions between sigma32 and DnaK
The three dimensional coordinates of the σ 32 -DnaK protein complex have been generated using molecular docking technique with the help of the software tool GRAMM. When σ 32 interacts with DnaK no significant changes occur in the σ 32 structure (r.m.s.d. between σ 32 in σ 32 -DnaK complex and σ 32 alone 0.2Å). Analysis of σ 32 -DnaK protein complex shows that the interactions between the proteins have been stabilized mainly by H-bonds involving the side chain atoms of the proteins. The residues involved in interactions are shown in Table 1 (see supplementary material) & (Figure 1 B). Interestingly, it has been observed that all the amino acid residues from σ 32 , involved in binding DnaK fall in the region2 and region3 of σ 32 . Though the aforementioned regions have previously been predicted to be involved in binding with DnaK and responsible for σ 32 specific functions [9, 10], the molecular details of the binding have not yet been established. This report elucidates the structural details of σ 32 as well as the interactions between σ 32 and DnaK. Heating σ 32 -DnaK protein complex beyond 42˚C, destroys all six H-bond and results in total loss of interaction between σ 32 and DnaK. This is because of the loss of structural arrangements in the binding interface of the σ 32 protein. This is exemplified by r.m.s.d value (8.276Å) of the backbone atoms of σ 32 protein above 43˚C (Figure 1 C). This might play a major role in decreasing the degradation rate of σ 32 . Major structural change has been identified to occur in the binding interface of σ 32 -DnaK protein complex. This result is consistent to earlier studies by Chattopadhyay et al. 2002, which reports that this abolition of σ 32 -DnaK interaction is mainly due to the structural changes of σ 32 at heat shock temperature.

Conclusion:
In this paper an attempt has been made to analyze the probable molecular details of the interactions of σ 32 with DnaK. The three dimensional structure of σ 32 has been predicted using the homology modeling technique. The functions of σ 32 are dependent on temperature. Therefore, the modeled structure of σ 32 has been heated from 32˚C to 43˚C. It revealed a huge structural change beyond 42˚C. The σ 32 is known to bind DnaK. In order to elucidate the mode of binding of σ 32 with DnaK, the three dimensional coordinates of the σ 32 and DnaK have been used to dock the two proteins together by molecular docking. The interaction scheme revealed that the role of amino acid residues from the region 2 and region 3 of σ 32 . This report deals with the detailed molecular biochemistry of temperature dependence of σ 32 structure and σ 32 -DnaK interactions. Therefore our study will be useful for future genetic studies to elucidate the roles of the amino acid residues in the proteinprotein interactions for the heat shock response.