Structural Analysis of Ino2p/Ino4p Mutual Interactions and Their Binding Interface with Promoter DNA.

Gene expression is mediated by a series of regulatory proteins, i.e., transcription factors. Under different growth conditions, the transcriptional regulation of structural genes is associated with the recognition of specific regulatory elements (REs) in promoter DNA. The manner by which transcription factors recognize distinctive REs is a key question in structural biology. Previous research has demonstrated that Ino2p/Ino4p heterodimer is associated with the transcriptional regulation of phospholipid biosynthetic genes. Mechanistically, Ino2p/Ino4p could specifically recognize the inositol/choline-responsive element (ICRE), followed by the transcription activation of the phospholipid biosynthetic gene. While the promoter DNA sequence for Ino2p has already been characterized, the structural basis for the mutual interaction between Ino2p/Ino4p and their binding interface with promoter DNA remain relatively unexplored. Here, we have determined the crystalline structure of the Ino2pDBD/Ino4pDBD/DNA ternary complex, which highlights some residues (Ino2pHis12/Glu16/Arg20/Arg44 and Ino4pHis12/Glu16/Arg19/Arg20) associated with the sequence-specific recognition of promoter DNA. Our biochemical analysis showed that mutating these residues could completely abolish protein-DNA interaction. Despite the requirement of Ino2p and Ino4p for interprotein-DNA interaction, both proteins can still interact-even in the absence of DNA. Combined with the structural analysis, our in vitro binding analysis demonstrated that residues (Arg35, Asn65, and Gln69 of Ino2pDBD and Leu59 of Ino4pDBD) are critical for interprotein interactions. Together, these results have led to the conclusion that these residues are critical to establishing interprotein-DNA and protein-DNA mutual interactions.

The two paralogous copies of the YoeB-YefM toxin-antitoxin module in Staphylococcus aureus differ in DNA binding and recognition patterns.

Toxin-antitoxin (TA) systems are ubiquitous regulatory modules for bacterial growth and cell survival following stress. YefM-YoeB, the most prevalent type II TA system, is present in a variety of bacterial species. In Staphylococcus aureus, the YefM-YoeB system exists as two independent paralogous copies. Our previous research resolved crystal structures of the two oligomeric states (heterotetramer and heterohexamer-DNA ternary complex) of the first paralog as well as the molecular mechanism of transcriptional autoregulation of this module. However, structural details reflecting molecular diversity in both paralogs have been relatively unexplored. To understand the molecular mechanism of how Sa2YoeB and Sa2YefM regulate their own transcription and how each paralog functions independently, we solved a series of crystal structures of the Sa2YoeB-Sa2YefM. Our structural and biochemical data demonstrated that both paralogous copies adopt similar mechanisms of transcriptional autoregulation. In addition, structural analysis suggested that molecular diversity between the two paralogs might be reflected in the interaction profile of YefM and YoeB and the recognition pattern of promoter DNA by YefM. Interaction analysis revealed unique conformational and activating force effected by the interface between Sa2YoeB and Sa2YefM. In addition, the recognition pattern analysis demonstrated that residues Thr7 and Tyr14 of Sa2YefM specifically recognizes the flanking sequences (G and C) of the promoter DNA. Together, these results provide the structural insights into the molecular diversity and independent function of the paralogous copies of the YoeB-YefM TA system.

Crystal structures of anthranilate phosphoribosyltransferase from Saccharomyces cerevisiae.

Anthranilate phosphoribosyltransferase (AnPRT) catalyzes the transfer of the phosphoribosyl group of 5'-phosphoribosyl-1'-pyrophosphate (PRPP) to anthranilate to form phosphoribosyl-anthranilate. Crystal structures of AnPRTs from bacteria and archaea have previously been determined; however, the structure of Saccharomyces cerevisiae AnPRT (ScAnPRT) still remains unsolved. Here, crystal structures of ScAnPRT in the apo form as well as in complex with its substrate PRPP and the substrate analogue 4-fluoroanthranilate (4FA) are presented. These structures demonstrate that ScAnPRT exhibits the conserved structural fold of type III phosphoribosyltransferase enzymes and shares the similar mode of substrate binding found across the AnPRT protein family. In addition, crystal structures of ScAnPRT mutants (ScAnPRTSer121Ala and ScAnPRTGly141Asn) were also determined. These structures suggested that the conserved residue Ser121 is critical for binding PRPP, while Gly141 is dispensable for binding 4FA. In summary, these structures improved the preliminary understanding of the substrate-binding mode of ScAnPRT and laid foundations for future research.

Distinct oligomeric structures of the YoeB-YefM complex provide insights into the conditional cooperativity of type II toxin-antitoxin system.

YoeB-YefM, the widespread type II toxin-antitoxin (TA) module, binds to its own promoter to autoregulate its transcription: repress or induce transcription under normal or stress conditions, respectively. It remains unclear how YoeB-YefM regulates its transcription depending on the YoeB to YefM TA ratio. We find that YoeB-YefM complex from S.aureus exists as two distinct oligomeric assemblies: heterotetramer (YoeB-YefM2-YoeB) and heterohexamer (YoeB-YefM2-YefM2-YoeB) with low and high DNA-binding affinities, respectively. Structures of the heterotetramer alone and heterohexamer bound to promoter DNA reveals that YefM C-terminal domain undergoes disorder to order transition upon YoeB binding, which allosterically affects the conformation of N-terminal DNA-binding domain. At TA ratio of 1:2, unsaturated binding of YoeB to the C-terminal regions of YefM dimer forms an optimal heterohexamer for DNA binding, and two YefM dimers with N-terminal domains dock into the adjacent major grooves of DNA to specifically recognize the 5'-TTGTACAN6AGTACAA-3' palindromic sequence, resulting in transcriptional repression. In contrast, at TA ratio of 1:1, binding of two additional YoeB molecules onto the heterohexamer induces the completely ordered conformation of YefM and disassembles the heterohexamer into two heterotetramers, which are unable to bind the promoter DNA optimally due to steric clashes, hence derepresses TA operon transcription.

Structural insights into the formation of oligomeric state by a type I Hsp40 chaperone.

Molecular chaperones can prevent and repair protein misfolding and aggregation to maintain protein homeostasis in cells. Hsp40 chaperones interact with unfolded client proteins via the dynamic multivalent interaction (DMI) mechanism with their multiple client-binding sites. Here we report that a type I Hsp40 chaperone from Streptococcus pneumonia (spHsp40) forms a concentration-independent polydispersity oligomer state in solution. The crystal structure of spHsp40 determined at 2.75 Å revealed that each monomer has a type I Hsp40 structural fold containing a zinc finger domain and C-terminal domains I and II (CTD I and CTD II). Subsequent quaternary structure analysis using a PISA server generated two dimeric models. The interface mutational analysis suggests the conserved C-terminal dimeric motif as a basis for dimer formation and that the novel dimeric interaction between a client-binding site in CTD I and the zinc finger domain promotes the formation of the spHsp40 oligomeric state. In vitro functional analysis demonstrated that spHsp40 oligomer is fully active and possess the optimal activity in stimulating the ATPase activity of spHsp70. The oligomer state of type I Hsp40 and its formation might be important in understanding Hsp40 function and its interaction with client proteins.

Crystallographic Analysis of the Catalytic Mechanism of Phosphopantothenoylcysteine Synthetase from Saccharomyces cerevisiae.

Phosphopantothenoylcysteine (PPC) synthetase (PPCS) catalyzes nucleoside triphosphate-dependent condensation reaction between 4'-phosphopantothenate (PPA) and l-cysteine to form PPC in CoA biosynthesis. The catalytic mechanism of PPCS has not been resolved yet. Coenzyme A biosynthesis protein 2 (Cab2) possesses activity of PPCS in Saccharomyces cerevisiae. Our enzymatic assays suggest that Cab2 could utilize both ATP and CTP to activate PPA in vitro. The results of isothermal titration calorimetry indicate that PPA, CTP, and ATP could bind to Cab2 individually, with PPA having the highest binding affinity. To provide further insight into the catalytic mechanism of Cab2, we determined the crystal structures of Cab2 and its complex with PPA, the reaction intermediate 4'-phosphopantothenoyl-CMP, the final reaction product PPC, and the product analogue phosphopantothenoylcystine. Except for PPA, all other ligands were generated in situ and present in the active-site pocket of Cab2. Structures of Cab2 in complex with ligands provide insight into substrates binding and its catalytic mechanism. Analysis of structures indicates that the carboxyl of PPA-moiety of ligands and the γ-amino group of Asn97 possess different conformations in these complex structures. The cysteine/cystine/serine selectivity assays for Cab2 indicate that the amino group rather than the thiol group of l-cysteine attacks the carbonyl of 4'-phosphopantothenoyl-CMP to form PPC. Based on structural and biochemical data, the catalytic mechanism of Cab2 was proposed for the first time.

The C-terminus of ubiquitin plays a critical role in deamidase Lpg2148 recognition.

By bearing a papain-like core structure and a cysteine-based catalytic triad, deamidase can convert glutamine to glutamic acid or asparagine to aspartic acid to modify the functions of host target proteins resulting in the blocking of eukaryotic host cell function. Legionella pneumophila effector Lpg2148 (MvcA) is a deamidase, a structural homolog of cycle inhibiting factor (Cif) effectors. Lpg2148 and Cif effectors are functionally diverse, with Lpg2148 only catalyzing ubiquitin but not NEDD8. However, a detailed understanding of substrate specificity is still missing. Here, we resolved the crystal structure of Lpg2148 at 2.5 Šresolution and obtained rigid-body modeling of Lpg2148 with C-terminus deleted ubiquitin (1-68) (ubΔc) complex using HADDOCK, which shows that the C-terminus of ubiquitin is flexible in recognition. We also conducted the truncated analysis to demonstrate that Leu71 of ubiquitin is necessary for its interaction with Lpg2148. Moreover, Val33 of Lpg2148 at the edge of a channel plays a vital role in the interaction and is limited by the length of the C-terminus of ubiquitin, which may help to explain the selectivity of ubiquitin over NEDD8. In summary, these results enrich our knowledge of substrate recognition of deamidase.