Michelle A. Markus

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.

Mechanistic pathways in CF3COOH-mediated deacetalization reactions.

It has been widely accepted that both the protection of carbonyls and the deprotection of acetals and ketals involve the participation of a water molecule: formation of acetals and ketals is a dehydration process, whereas the deprotection is often referred to as hydrolysis, which, as implied by its name, always requires the presence of water. Herein, we report experimental evidence and mechanistic investigations that provide an alternative view to this process. We have demonstrated that water is not required to convert acetals and ketals to the corresponding carbonyls. The (1)H NMR experimental results revealed that the TFA-mediated transformation of acetal to aldehyde occurs via a hemiacetal TFA ester intermediate, which differentiates itself from the classic acid-catalyzed hydrolysis, where the hemiacetal is the putative intermediate responsible for the formation of the aldehyde. More interestingly, alcohols are not the final byproducts as they are in the classical hydrolysis, rather, the two alcohol molecules are converted to two TFA esters under the reaction conditions. On the basis of the NMR evidence, we have proposed that the two TFA esters are formed in two separate steps via a different mechanism along the reaction pathway. Formation of the TFA esters renders the reaction irreversible. To the best of our knowledge, the cascade reaction pathway presented by the TFA-mediated conversion of acetals and ketals to carbonyls has never been previously postulated.

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.

1H, 15N, 13C and 13CO resonance assignments and secondary structure of villin 14T, a domain conserved among actin-severing proteins

SummarySequence-specific assignments have been made for the 1H, 15N, 13C and 13CO resonances of 14T, the 126-residue amino-terminal domain of the actin-severing protein villin. Villin is a member of a family of proteins that regulate cytoskeletal actin by severing, capping and nucleating actin filaments. Actin binding is dependent on calcium and disrupted by phosphatidyl inositol 4,5-bisphosphate. Actin-severing proteins are built from three or six repeats of a conserved domain, represented by 14T. Expression in Escherichia coli facilitated incorporation of 15N and 13C isotopes and application of triple-resonance, backbone-directed strategies for the sequential assignments. Elements of regular secondary structure have been identified by characteristic patterns of NOE cross peaks and values of vicinal 3JHnHα coupling constants. Amide protons that exchange slowly (rates less than 1.0×10-4 per min) are concentrated in the central β-sheet and the second and third α-helices, suggesting that these elements of secondary structure form very stable hydrogen bonds. Assignments for the amide nitrogens and protons have been examined as a function of pH and calcium concentration. Based on the conservation of chemical shifts in the core of the domain, villin 14T maintains the same overall fold in the pH range from 4.15 to 6.91 and the calcium range from 0 to 50 mM. The calcium data indicate the presence of two calcium-binding sites and suggest their locations.

Solution structure of wild-type human matrix metalloproteinase 12 (MMP-12) in complex with a tight-binding inhibitor.

MMP-12 (macrophage elastase) is a member of a family of proteases that target proteins of the extracellular matrix. Matrix metalloproteinases (MMPs) influence tissue remodeling both in healthy growth and development and in the progression of pathological conditions such as arthritis and cancer. Members of the MMP family are specific for different components of the extracellular matrix and thus contribute to different disease states. They share a strongly conserved active site, including a catalytic zinc ion, coordinated by three conserved histidine side chains. The active site is contained within a groove along one face of the protein, the substrate binding cleft, that accommodates the protein chain to be cleaved. One wall of the substrate binding cleft is formed by the specificity loop, the loop between helix B and helix C. This loop varies in composition and length among members of the MMP family and contributes to the substrate specificity of the individual MMPs by forming much of the S10 binding pocket. MMP-12 was identified as the elastase activity produced by alveolar macrophages (Shapiro et al. 1993), although it shows activity against a range of basement membrane components, including fibronectin, laminin, entactin, chondroitan sulfate, and heparin sulfate (Gronski et al. 1997). Patients suffering from chronic obstructive pulmonary disease (COPD) show higher levels of MMP-12 in lung tissue and bronchoalveolar lavage (Molet et al. 2005). MMP-12 knockout mice are resistant to emphysema even when exposed to cigarette smoke (Hautamaki et al. 1997). These observations suggest that an MMP-12 inhibitor is a potentially disease-modifying treatment for COPD. Previous MMP inhibitors have been plagued with musculoskeletal side effects (Wojtowicz-Praga 1998), presumably due to a lack of selectivity for the target enzyme. For many compounds, this lack of selectivity can be traced to a hydroxamate functionality, which provides affinity by chelating the catalytic zinc ion but prevents specificity, since all the enzymes in this family contain this zinc. To avoid these specificity issues, we have been interested in developing an inhibitor lacking the M. A. Markus (&) B. Dwyer S. Wolfrom K. Malakian J. Wilhelm D. H. H. Tsao Structural Biology and Computational Chemistry, Chemical and Screening Sciences, Wyeth Research, 87 CambridgePark Drive, Cambridge, MA 02140, USA e-mail: mmarkus@wyeth.com

Solving the atomic resolution structure of a protein in solution: nuclear magnetic resonance studies of villin 14T

To understand how a protein functions, it is essential to know the three-dimensional structure of the protein to atomic resolution. Multidimensional nuclear magnetic resonance (NMR) techniques provide one method for solving atomic resolution protein structures. These techniques have been applied to the 126-residue protein domain, villin 14T. The most challenging step is assigning each resonance line in the NMR spectrum to the correct proton within the protein. For villin 14T, this sequential assignment step was accomplished with triple-resonance, backbone-directed strategies. The structure reveals a unique fold shared only by domains from other proteins in the actin-severing family.