Early Work

Research in the Schepartz laboratory focuses on the chemical biology of protein–protein and protein–DNA interactions inside the cell. We are interested in the structural and energetic factors that distinguish specific protein interfaces and how the assembly (and disassembly) of these complexes define biology.

Specific questions our lab focuses on include how cells effectively use a limited number of transcription factors to achieve a precisely controlled and robust gene-regulatory network, how this network is usurped when cells succumb to viral attack, and how, inspired by the viral hijackers, we can design miniature proteins that mimic (and sometimes surpass) the functional properties of proteins found in Nature.

For a description of very early work in the Schepartz lab, click here.

We are very interested in understanding the detailed mechanisms by which transcription factors identify their preferred target sites within a sea of genomic DNA and unrelated protein. These studies began with Bernard Cuenoud’s Science report of a series of molecules in which the relative orientations of two peptides from the DNA-binding region of GCN4 (a basic region-leucine zipper transcription factor) were varied with a structurally tunable metal complex.    The properties of these molecules suggested that the sequence specificities of bZIP proteins might depend on the orientations of their DNA-binding regions.

Steven Metallo later verified this hypothesis, which represented a fundamentally new mechanism for controlling the sequence specificity of DNA-binding proteins.

Another novel mechanism for controlling sequence specificity was reported by David Paolella in Science. He discovered that an intrinsic bend could pre-organize DNA for binding by some proteins and not others. 

Steven Metallo and Jennifer Kohler recently described a third mechanism by which specificity — in this case, kinetic specificity — can be achieved.

They discovered that many bZIP and bHLHzip proteins bind DNA as monomers, dimerizing while DNA-bound, and that this monomer-binding pathway represents the most rapid search method for locating specific DNA.  

Our group is also very interested in how bZIP proteins are recognized and discriminated by viral proteins that hijack cellular transcriptional machinery for viral replication. Anne Baranger reported in Nature that the HTLV-I Tax protein increases the affinity of a cellular bZIP protein for viral DNA by altering the specificity of DNA binding, not by enhancing bZIP dimerization.  Subsequent work by Rodgers Palmer demonstrated that the HBV X protein employs an identical mechanism, despite the evolutionary distance between HBV and HTLV-I and the lack of sequence homology between Tax and pX. Though viral proteins have evolved to commandeer cellular transcription factors for viral replication, the resultant side effects on cellular transcription are responsible for producing diseased states. Tanya Schneider showed that pX diminishes the inherent specificity of the monomer-binding pathway of bZIPs, providing a molecular explanation for aberrant transcriptional activation during HBV infection.  Most recently, Mary Kay Pflum reported that pX bypasses the need for phosphorylation to activate certain bZIP proteins, short-circuiting a natural signal transduction pathway linking elevated cAMP levels and transcriptional activation. Her work provides a direct link between HBV infection and the development of hepatocellular carcinoma. 

Guided creatively by insights gleaned from our work on Tax and pX, our most recent efforts focus on the design of molecules that bind protein surfaces and inhibit protein-protein interactions inside the cell. Our interest in this area stems from the over-arching challenge and opportunity posed by the sequencing of the human genome. The actions and interactions of the proteins encoded by these 35,000 genes — whose numbers could reach 350,000 or more when post-translational modifications, alternative splicing, and proteolytic cleavage are considered — determine the full range of cellular activities in normal cell processes and in disease.

Our laboratory has described a general solution to the design of molecules capable of deconvoluting the role of transient protein–protein interactions inside the cell. Building on the observation that proteins generally recognize each other using extended, shallow, hydrophilic interfaces, we proposed that the most appropriate ligands for protein surfaces might be a miniature (but well folded) protein that presents a preorganized functional epitope in a very small package. Initially Neal Zondlo and Jason Chin reported in JACS     the successful use of protein grafting in the design and evolution of DNA-binding miniature proteins based on the structure of GCN4.

These molecules, containing only 36 residues, bind specific DNA under physiological conditions with comparable affinities and higher specificities that GCN4 itself. This work was highlighted in both Science and Nature. More recently Jin Montclare reported in JACS that it is possible to miniaturize both the recognition surface and the structural framework of a globular protein fold through the design of a miniature homeodomain that binds specific DNA without benefit of an “N-terminal arm.”  Jason Chin most recently has translated this research to the area of protein recognition by the design and discovery of miniature proteins that bind with nanomolar affinity to the anti-apoptotic proteins Bcl-2 and Bcl-XL.    Stacey Rutledge and Heather Volkman have shown that miniature proteins possessing the elusive goal of high affinity and specificity can be achieved even in cases where the binding site is exceptionally featureless and when the ligand is phosphorylated. Dasantila Golemi has shown that miniature proteins posessing paralog specificity can be designed de novo and can distinguish members of a protein family that are 60 percent identical and structurally superimposable. The newest miniature proteins use the PPII helix to distinguish between EVH1-domains and induce a unique phenotype in mammalian cells.

The observation that miniature protein ligands achieve the elusive goal of high affinity and (even more importantly) specificity suggests that could have enormous utility as probes of protein–protein interactions inside the cell, achieving a phenotypic precision that is difficult to achieve any other way. Many current projects in our lab are focused in this area and in the use of proteome chips to evaluate and aid miniature protein design.