The traditional view of protein-ligand binding treats a protein as comprising distinct binding epitopes on the surface of a degenerate structural scaffold, largely ignoring the impact of a protein’s energy landscape. To determine the robustness of this simplification, we compared two small helix-turn-helix transcription factors with different energy landscapes. λ-repressor is stable and well folded, while MarA appears to be marginally stable with multiple native conformations (molten). While λ-repressor is known to tolerate any hydrophobic mutation in the core, we find MarA drastically less tolerant to core mutation. Moreover, core mutations in MarA (distant from the DNA-binding interface) change the relative affinities of its binding partners, altering ligand specificity. These results can be explained by taking into account the effects of mutations on the entire energy landscape and not just the native state. Thus, for proteins with multiple conformations that are close in energy, such as many intrinsically disordered proteins, residues distant from the active site can alter both binding affinity and specificity.

Understanding the nature of partially folded intermediates transiently populated during protein folding is important for understanding both protein folding and misfolding. These ephemeral species, however, often elude direct experimental characterization. The well-characterized protein ribonuclease H from E. coli populates an on-pathway intermediate identified in both bulk studies and single molecule mechanical unfolding experiments. Here, we set out to trap the transient intermediate of RNase H at equilibrium by selectively destabilizing the region of the protein known to be unfolded in this species. Surprisingly, a single change at Ile 25 (I25A) resulted in the equilibrium population of the intermediate under near-native conditions. The intermediate was undetectable in a series of HSQC's, revealing the dynamic nature of this partially unfolded form on the timescale of NMR detection. This result is in contrast to studies in which the structures of trapped intermediates are solved by NMR, indicating that the they are well-packed and native-like. The dynamic nature of the RNase H intermediate may be important for its role as an on-pathway, productive species that promotes efficient folding.Partially folded intermediates are known to play an important role in the mechanism of protein folding. For some proteins, intermediates appear to play a productive role, aiding in the formation of the native fold, while for others they constitute an off-pathway species. In addition to their importance in folding mechanisms, partially folded forms may also play crucial biological roles. There are many examples of proteins with unstructured regions whose disordered-ordered folding transitions are important for binding and other regulatory events 1; 2 . For example, the active form of the steroidogenic acute regulatory protein appears to be a molten globule 3 , a state usually associated with early folding intermediates. Such intermediates have also been implicated in the formation of aggregation-prone species 4 . In spite of their clear importance in both folding and function, these partially folded intermediates are usually not amenable to detailed structural and biophysical studies due to their low populations at equilibrium and transient nature.Here, we set out to trap the intermediate of ribonuclease H from E. coli (RNase H), a small (17.5 kDa), monomeric enzyme known to fold through such a partially folded kinetic 1 To whom correspondence should be addressed: marqusee@berkeley.edu or phone: 510-642-7678. * These authors contributed equally to this work # Department of Molecular & Cell Biology ± Chemical Biology Graduate Group, Department of Chemistry Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production p...

Supplementary Table from The Fibronectin–ILT3 Interaction Functions as a Stromal Checkpoint that Suppresses Myeloid Cells

Supplementary Figure from The Fibronectin–ILT3 Interaction Functions as a Stromal Checkpoint that Suppresses Myeloid Cells

Supplementary Table from The Fibronectin–ILT3 Interaction Functions as a Stromal Checkpoint that Suppresses Myeloid Cells

<div>Abstract<p>Suppressive myeloid cells inhibit antitumor immunity by preventing T-cell responses. Immunoglobulin-like transcript 3 (ILT3; also known as LILRB4) is highly expressed on tumor-associated myeloid cells and promotes their suppressive phenotype. However, the ligand that engages ILT3 within the tumor microenvironment and renders tumor-associated myeloid cells suppressive is unknown. Using a screening approach, we identified fibronectin as a functional ligand for ILT3. The interaction of fibronectin with ILT3 polarized myeloid cells toward a suppressive state, and these effects were reversed with an ILT3-specific antibody that blocked the interaction of ILT3 with fibronectin. Furthermore, <i>ex vivo</i> treatment of human tumor explants with anti-ILT3 reprogrammed tumor-associated myeloid cells toward a stimulatory phenotype. Thus, the ILT3–fibronectin interaction represents a “stromal checkpoint” through which the extracellular matrix actively suppresses myeloid cells. By blocking this interaction, tumor-associated myeloid cells may acquire a stimulatory phenotype, potentially resulting in increased antitumor T-cell responses.</p></div>

No abstract

<div>Abstract<p>Suppressive myeloid cells inhibit antitumor immunity by preventing T-cell responses. Immunoglobulin-like transcript 3 (ILT3; also known as LILRB4) is highly expressed on tumor-associated myeloid cells and promotes their suppressive phenotype. However, the ligand that engages ILT3 within the tumor microenvironment and renders tumor-associated myeloid cells suppressive is unknown. Using a screening approach, we identified fibronectin as a functional ligand for ILT3. The interaction of fibronectin with ILT3 polarized myeloid cells toward a suppressive state, and these effects were reversed with an ILT3-specific antibody that blocked the interaction of ILT3 with fibronectin. Furthermore, <i>ex vivo</i> treatment of human tumor explants with anti-ILT3 reprogrammed tumor-associated myeloid cells toward a stimulatory phenotype. Thus, the ILT3–fibronectin interaction represents a “stromal checkpoint” through which the extracellular matrix actively suppresses myeloid cells. By blocking this interaction, tumor-associated myeloid cells may acquire a stimulatory phenotype, potentially resulting in increased antitumor T-cell responses.</p></div>