Crystallization and preliminary X-ray diffraction studies of the metal-ion-mediated ternary complex of the HutP protein with L-histidine and its cognate RNA.

HutP is an RNA-binding protein that regulates the expression of the Bacillus subtilis hut operon by binding to cis-acting regulatory sequences within hut mRNA, exclusively in the presence of L-histidine. We recently solved the crystal structure of a binary complex (HutP with an L-histidine analog) that revealed a novel RNA-binding fold, and identified the important residues that interact with the L-histidine analog. In addition, we have defined the minimal RNA binding segment that is required for HutP recognition. Interestingly, we showed that ternary complex formation depends on the availability of not only L-histidine but also divalent metal ions. Here we report the crystallization and preliminary X-ray diffraction analysis of the HutP ternary complex. The ternary complex was crystallized in the presence of Mg2+ along with L-histidine and hut mRNA, using the hanging drop vapor diffusion method. The crystal belongs to the R3 space group, with unit cell parameters a=b=75.30 A, c=133.8 A. A complete data set at 1.60 A was collected.

Identification of important chemical groups of the hut mRNA for HutP interactions that regulate the hut operon in Bacillus subtilis.

HutP is an RNA binding protein that regulates the expression of the histidine utilization (hut) operon in Bacillus species by binding to cis-acting regulatory sequences on hut mRNA. We recently solved the HutP crystal structure, which revealed a novel fold where three dimers are arranged in a 3-fold axis to form the hexamer. We also identified a minimal RNA binding element sufficient for HutP binding: three UAG trinucleotide motifs, each separated by 4 nt, located just upstream of the terminator. In the present study we have identified important RNA chemical groups essential for HutP interactions, by combining an in vitro selection strategy and analyses by site-specific base substitutions. These analyses suggest that each HutP molecule recognizes one UAG motif, where the first base (U) can be substituted with other bases, while the second and third bases (A and G) are required for the interactions. Further analyses of the chemical groups of the A and G bases in the UAG motif by modified base analogs suggested the importance of the exocyclic NH2 group in these bases. Also, in this motif, only the 2'-OH group of A is important for HutP recognition. Considering the important chemical groups identified here, as well as the electrostatic potential analysis of HutP, we propose that Glu137 is one of the important residues for the HutP-RNA interactions.

Influence of medium- and long-range interactions in different folding types of globular proteins.

Recognition of protein fold from amino acid sequence is a challenging task. The structure and stability of proteins from different fold are mainly dictated by inter-residue interactions. In our earlier work, we have successfully used the medium- and long-range contacts for predicting the protein folding rates, discriminating globular and membrane proteins and for distinguishing protein structural classes. In this work, we analyze the role of inter-residue interactions in commonly occurring folds of globular proteins in order to understand their folding mechanisms. In the medium-range contacts, the globin fold and four-helical bundle proteins have more contacts than that of DNA-RNA fold although they all belong to all-alpha class. In long-range contacts, only the ribonuclease fold prefers 4-10 range and the other folding types prefer the range 21-30 in alpha/beta class proteins. Further, the preferred residues and residue pairs influenced by these different folds are discussed. The information about the preference of medium- and long-range contacts exhibited by the 20 amino acid residues can be effectively used to predict the folding type of each protein.

Crystallization and preliminary X-ray diffraction studies of HutP protein: an RNA-binding protein that regulates the transcription of hut operon in Bacillus subtilis.

HutP is an RNA-binding protein and regulates the expression of the histidine utilization (hut) operon in Bacillus subtilis by binding to cis-acting regulatory sequences on hut mRNA. HutP and its mutant, which has increased affinity for the regulatory sequences, were purified and crystallized by the hanging-drop vapor diffusion method. The space group was P2(1)3 with unit cell dimensions a=b=c=95.6A for HutP and a=b=c=96.8A for the mutant. Complete data sets of 3.0-A resolution for wild-type HutP and of 2.70-A resolution for the mutant HutP were collected.

Distribution of amino acid residues and residue-residue contacts in molecular chaperones.

The amino acid distribution and residue-residue contacts in molecular chaperones are different when compared to normal globular proteins. The study of molecular chaperones reveals a different surrounding environment to exist for the residues Cys, Trp, and His which may play an important role in determining the chaperone structures. Unlike globular proteins, it has been observed that a one-to-one correspondence between the amino acid distribution in a sequence and the structures of molecular chaperones. The preference of amino acid residues surrounding all 20 types of residues in secondary structures and their accessible surface areas have been analysed.

Structural class prediction: an application of residue distribution along the sequence.

Deciphering the native conformation of proteins from their amino acid sequences is one of the most challenging problems in molecular biology. Information on the secondary structure of a protein can be helpful in understanding its native folded state. In our earlier work on molecular chaperones, we have analyzed the hydrophobic and charged patches, short-, medium- and long-range contacts and residue distributions along the sequence. In this article, we have made an attempt to predict the structural class of globular and chaperone proteins based on the information obtained from residue distributions. This method predicts the structural class with an accuracy of 93 and 96%, respectively, for the four- and three-state models in a training set of 120 globular proteins, and 90 and 96%, respectively, for a test set of 80 proteins. We have used this information and methodology to predict the structural classes of chaperones. Interestingly most of the chaperone proteins are predicted under alpha/beta or mixed folding type.

Analysis of hydrophobic and charged patches and influence of medium- and long-range interactions in molecular chaperones.

The amino acid composition of the aromatic residues Phe, Tyr and Trp are much less significant in chaperones and the residues Cys, Glu, His, Met and Pro vary significantly in chaperones compared to normal globular proteins. In the present work, we have analysed the hydrophobic and charged patches in molecular chaperones which provide more insight for a better understanding of chaperone folding. Also, we have investigated the role of medium- and long-range contacts in chaperones and the preference of amino acid residues influenced by these interactions. Furthermore, the role of hydrophobic and helix-forming residues and disulfide bonding in these interactions have been discussed.

Solvent accessibility analysis on the mutants of Hsc70 ATPase fragment.

Molecular chaperones are the cellular proteins which mediate the correct folding of other polypeptides. The concept of 'solvent accessibility' is one of the most powerful tools to understand the structure and stability of protein molecules. The hydrophobic variation of amino acid residues due to point mutations at many active sites of chaperone protein Hsc70 using solvent accessibility analysis is carried out. The numerical indices for several properties of amino acid residues, such as, reduction in accessibility, preference of amino acid residues in interior and surface parts, transfer free energy and the preference of amino acid residues to change their positions (buried/exposed) due to amino acid substitutions for Hsc70 and its mutants were set up. The accessibility of amino acid residues varies much between native and mutant proteins whereas there is no major changes on their conformations. The conformational stability for Hsc70 and its mutants were established and the computed hydrophobic free energy change is around 10 kcal/mol due to single amino acid substitution.