Barak Akabayov

The C-terminal domain of eukaryotic initiation factor 5 promotes start codon recognition by its dynamic interplay with eIF1 and eIF2β.

Recognition of the proper start codon on mRNAs is essential for protein synthesis, which requires scanning and involves eukaryotic initiation factors (eIFs) eIF1, eIF1A, eIF2, and eIF5. The carboxyl terminal domain (CTD) of eIF5 stimulates 43S preinitiation complex (PIC) assembly; however, its precise role in scanning and start codon selection has remained unknown. Using nuclear magnetic resonance (NMR) spectroscopy, we identified the binding sites of eIF1 and eIF2β on eIF5-CTD and found that they partially overlapped. Mutating select eIF5 residues in the common interface specifically disrupts interaction with both factors. Genetic and biochemical evidence indicates that these eIF5-CTD mutations impair start codon recognition and impede eIF1 release from the PIC by abrogating eIF5-CTD binding to eIF2β. This study provides mechanistic insight into the role of eIF5-CTDs dynamic interplay with eIF1 and eIF2β in switching PICs from an open to a closed state at start codons.

Impact of macromolecular crowding on DNA replication

Enzymatic activities in vivo occur in a crowded environment composed of many macromolecules. This environment influences DNA replication by increasing the concentration of the constituents, desolvation, decreasing the degrees of freedom for diffusion and hopping of proteins onto DNA, and enhancing binding equilibria and catalysis. However, the effect of macromolecular crowding on protein structure is poorly understood. Here we examine macromolecular crowding using the replication system of bacteriophage T7 and we show that it affects several aspects of DNA replication; the activity of DNA helicase increases and the sensitivity of DNA polymerase to salt is reduced. We also demonstrate, using SAXS analysis, that the complex between DNA helicase and DNA polymerase/trx is far more compact in a crowded environment. The highest enzymatic activity corresponds to the most compact structure. Better knowledge of the effect of crowding on structure and activity will enhance mechanistic insight beyond information obtained from NMR and X-ray structures.

Key feature of the catalytic cycle of TNF-α converting enzyme involves communication between distal protein sites and the enzyme catalytic core

Despite their key roles in many normal and pathological processes, the molecular details by which zinc-dependent proteases hydrolyze their physiological substrates remain elusive. Advanced theoretical analyses have suggested reaction models for which there is limited and controversial experimental evidence. Here we report the structure, chemistry and lifetime of transient metal–protein reaction intermediates evolving during the substrate turnover reaction of a metalloproteinase, the tumor necrosis factor-α converting enzyme (TACE). TACE controls multiple signal transduction pathways through the proteolytic release of the extracellular domain of a host of membrane-bound factors and receptors. Using stopped-flow x-ray spectroscopy methods together with transient kinetic analyses, we demonstrate that TACEs catalytic zinc ion undergoes dynamic charge transitions before substrate binding to the metal ion. This indicates previously undescribed communication pathways taking place between distal protein sites and the enzyme catalytic core. The observed charge transitions are synchronized with distinct phases in the reaction kinetics and changes in metal coordination chemistry mediated by the binding of the peptide substrate to the catalytic metal ion and product release. Here we report key local charge transitions critical for proteolysis as well as long sought evidence for the proposed reaction model of peptide hydrolysis. This study provides a general approach for gaining critical insights into the molecular basis of substrate recognition and turnover by zinc metalloproteinases that may be used for drug design.

Conformational dynamics of bacteriophage T7 DNA polymerase and its processivity factor, Escherichia coli thioredoxin

Gene 5 of bacteriophage T7 encodes a DNA polymerase (gp5) responsible for the replication of the phage DNA. Gp5 polymerizes nucleotides with low processivity, dissociating after the incorporation of 1 to 50 nucleotides. Thioredoxin (trx) of Escherichia coli binds tightly (Kd = 5 nM) to a unique segment in the thumb subdomain of gp5 and increases processivity. We have probed the molecular basis for the increase in processivity. A single-molecule experiment reveals differences in rates of enzymatic activity and processivity between gp5 and gp5/trx. Small angle X-ray scattering studies combined with nuclease footprinting reveal two conformations of gp5, one in the free state and one upon binding to trx. Comparative analysis of the DNA binding clefts of DNA polymerases and DNA binding proteins show that the binding surface contains more hydrophobic residues than other DNA binding proteins. The balanced composition between hydrophobic and charged residues of the binding site allows for efficient sliding of gp5/trx on the DNA. We propose a model for trx-induced conformational changes in gp5 that enhance the processivity by increasing the interaction of gp5 with DNA.

Using softer X-ray absorption spectroscopy to probe biological systems

Many inorganic species are now recognized as being essential for life, including many forms of sulfur, phosphate and numerous classes of metal ions. For example, recent progress in the fields of biochemistry and biology has pointed out the critical importance of sulfur in the biosynthesis of vital cofactors and active sites in proteins, and in the complex reaction mechanisms often involved. Special attention has also been drawn to the diverse roles of alkaline (Na(+), K(+)) and alkaline earth (Mg(2+), Ca(2+)) metal ions in mediating the activity of RNA, proteins and many processes in living cells. While the general effect of these ions in biology is mostly understood, information on their detailed role is deficient. Here the application of softer X-ray absorption spectroscopy (XAS) to probe the local structural and electronic environment of such ions within their biological complexes and during physiological reactions is discussed. In addition, the required experimental set-up and the difficulties associated with conducting softer XAS experiments on biological samples are presented.

Molecular Crowding Enhanced ATPase Activity of the RNA Helicase eIF4A Correlates with Compaction of Its Quaternary Structure and Association with eIF4G

Enzymatic reactions occur in a crowded and confined environment in vivo, containing proteins, RNA and DNA. Previous reports have shown that interactions between macromolecules, and reactions rates differ significantly between crowded environments and dilute buffers. However, the direct effect of crowding on the level of high-resolution structures of macromolecules has not been extensively analyzed and is not well understood. Here we analyze the effect of macromolecular crowding on structure and function of the human translation initiation factors eIF4A, a two-domain DEAD-Box helicase, the HEAT-1 domain of eIF4G, and their complex. We find that crowding enhances the ATPase activity of eIF4A, which correlates with a shift to a more compact structure as revealed with small-angle X-ray scattering. However, the individual domains of eIF4A, or the eIF4G-HEAT-1 domain alone show little structural changes due to crowding except for flexible regions. Thus, the effect of macromolecular crowding on activity and structure need to be taken into account when evaluating enzyme activities and structures of multidomain proteins, proteins with flexible regions, or protein complexes obtained by X-ray crystallography, NMR, or other structural methods.

An Interaction between DNA Polymerase and Helicase Is Essential for the High Processivity of the Bacteriophage T7 Replisome

Background: Interactions of DNA polymerase and DNA helicase are crucial in DNA synthesis. Results: Two distinct interactions are involved in formation of the DNA polymerase/DNA helicase complex. Conclusion: The multiple interactions between DNA polymerase and DNA helicase account for the high processivity of leading strand synthesis. Significance: Understanding of the replication process in bacteriophage T7 facilitates studies in more complex systems. Synthesis of the leading DNA strand requires the coordinated activity of DNA polymerase and DNA helicase, whereas synthesis of the lagging strand involves interactions of these proteins with DNA primase. We present the first structural model of a bacteriophage T7 DNA helicase-DNA polymerase complex using a combination of small angle x-ray scattering, single-molecule, and biochemical methods. We propose that the protein-protein interface stabilizing the leading strand synthesis involves two distinct interactions: a stable binding of the helicase to the palm domain of the polymerase and an electrostatic binding of the carboxyl-terminal tail of the helicase to a basic patch on the polymerase. DNA primase facilitates binding of DNA helicase to ssDNA and contributes to formation of the DNA helicase-DNA polymerase complex by stabilizing DNA helicase.

Isolation, Characterization, and Aggregation of a Structured Bacterial Matrix Precursor

Background: TasA is an extracellular matrix protein that makes amyloid-like fibers in Bacillus subtilis biofilms. Results: An isolated TasA matrix precursor self-assembled in vitro into fibers on hydrophobic surfaces and in acidic solutions. Conclusion: TasA is purified as stable, structured oligomers that aggregate in response to simple physical external cues. Significance: TasA aggregation principles can be used to design new anti-biofilm drugs and surfaces. Biofilms are surface-associated groups of microbial cells that are embedded in an extracellular matrix (ECM). The ECM is a network of biopolymers, mainly polysaccharides, proteins, and nucleic acids. ECM proteins serve a variety of structural roles and often form amyloid-like fibers. Despite the extensive study of the formation of amyloid fibers from their constituent subunits in humans, much less is known about the assembly of bacterial functional amyloid-like precursors into fibers. Using dynamic light scattering, atomic force microscopy, circular dichroism, and infrared spectroscopy, we show that our unique purification method of a Bacillus subtilis major matrix protein component results in stable oligomers that retain their native α-helical structure. The stability of these oligomers enabled us to control the external conditions that triggered their aggregation. In particular, we show that stretched fibers are formed on a hydrophobic surface, whereas plaque-like aggregates are formed in solution under acidic pH conditions. TasA is also shown to change conformation upon aggregation and gain some β-sheet structure. Our studies of the aggregation of a bacterial matrix protein from its subunits shed new light on assembly processes of the ECM within bacterial biofilms.

The interaction between eukaryotic initiation factor 1A and eIF5 retains eIF1 within scanning preinitiation complexes.

Scanning of the mRNA transcript by the preinitiation complex (PIC) requires a panel of eukaryotic initiation factors, which includes eIF1 and eIF1A, the main transducers of stringent AUG selection. eIF1A plays an important role in start codon recognition; however, its molecular contacts with eIF5 are unknown. Using nuclear magnetic resonance, we unveil eIF1As binding surface on the carboxyl-terminal domain of eIF5 (eIF5-CTD). We validated this interaction by observing that eIF1A does not bind to an eIF5-CTD mutant, altering the revealed eIF1A interaction site. We also found that the interaction between eIF1A and eIF5-CTD is conserved between humans and yeast. Using glutathione S-transferase pull-down assays of purified proteins, we showed that the N-terminal tail (NTT) of eIF1A mediates the interaction with eIF5-CTD and eIF1. Genetic evidence indicates that overexpressing eIF1 or eIF5 suppresses the slow growth phenotype of eIF1A-NTT mutants. These results suggest that the eIF1A-eIF5-CTD interaction during scanning PICs contributes to the maintenance of eIF1 within the open PIC.

DNA Recognition by the DNA Primase of Bacteriophage T7 : A Structure-Function Study of the Zinc-Binding Domain

Synthesis of oligoribonucleotide primers for lagging-strand DNA synthesis in the DNA replication system of bacteriophage T7 is catalyzed by the primase domain of the gene 4 helicase-primase. The primase consists of a zinc-binding domain (ZBD) and an RNA polymerase (RPD) domain. The ZBD is responsible for recognition of a specific sequence in the ssDNA template whereas catalytic activity resides in the RPD. The ZBD contains a zinc ion coordinated with four cysteine residues. We have examined the ligation state of the zinc ion by X-ray absorption spectroscopy and biochemical analysis of genetically altered primases. The ZBD of primase engaged in catalysis exhibits considerable asymmetry in coordination to zinc, as evidenced by a gradual increase in electron density of the zinc together with elongation of the zinc-sulfur bonds. Both wild-type primase and primase reconstituted from purified ZBD and RPD have a similar electronic change in the level of the zinc ion as well as the configuration of the ZBD. Single amino acid replacements in the ZBD (H33A and C36S) result in the loss of both zinc binding and its structural integrity. Thus the zinc in the ZBD may act as a charge modulation indicator for the surrounding sulfur atoms necessary for recognition of specific DNA sequences.