Parallel evolution of ligand specificity between LacI/GalR family repressors and periplasmic sugar-binding proteins

The bacterial LacI/GalR family repressors such as lactose operon repressor (LacI), purine nucleotide synthesis repressor (PurR), and trehalose operon repressor (TreR) consist of not only the N-terminal helix-turn-helix DNA-binding domain but also the C-terminal ligand-binding domain that is structurally homologous to periplasmic sugar-binding proteins. These structural features imply that the repressor family evolved by acquiring the DNA-binding domain in the N-terminal of an ancestral periplasmic binding protein (PBP). Phylogenetic analysis of the LacI/GalR family repressors and their PBP homologues revealed that the acquisition of the DNA-binding domain occurred first in the family, and ligand specificity then evolved. The phylogenetic tree also indicates that the acquisition occurred only once before the divergence of the major lineages of eubacteria, and that the LacI/GalR and the PBP families have since undergone extensive gene duplication/loss independently along the evolutionary lineages. Multiple alignments of the repressors and PBPs furthermore revealed that repressors and PBPs with the same ligand specificity have the same or similar residues in their binding sites. This result, together with the phylogenetic relationship, demonstrates that the repressors and the PBPs individually acquired the same ligand specificity by homoplasious replacement, even though their genes are encoded in the same operon.     

Allosteric Communication Occurs via Networks of Tertiary and Quaternary Motions in Proteins

Allosteric proteins bind an effector molecule at one site resulting in a functional change at a second site. We hypothesize that allosteric communication in proteins relies upon networks of quaternary (collective, rigid-body) and tertiary (residue–residue contact) motions. We argue that cyclic topology of these networks is necessary for allosteric communication. An automated algorithm identifies rigid bodies from the displacement between the inactive and the active structures and constructs “quaternary networks” from these rigid bodies and the substrate and effector ligands. We then integrate quaternary networks with a coarse-grained representation of contact rearrangements to form “global communication networks” (GCNs). The GCN reveals allosteric communication among all substrate and effector sites in 15 of 18 multidomain and multimeric proteins, while tertiary and quaternary networks exhibit such communication in only 4 and 3 of these proteins, respectively. Furthermore, in 7 of the 15 proteins connected by the GCN, 50% or more of the substrate-effector paths via the GCN are “interdependent” paths that do not exist via either the tertiary or the quaternary network. Substrate-effector “pathways” typically are not linear but rather consist of polycyclic networks of rigid bodies and clusters of rearranging residue contacts. These results argue for broad applicability of allosteric communication based on structural changes and demonstrate the utility of the GCN. Global communication networks may inform a variety of experiments on allosteric proteins as well as the design of allostery into non-allosteric proteins.     

Subdividing repressor function: DNA binding affinity, selectivity, and allostery can be altered by amino acid substitution of nonconserved residues in a LacI/GalR homologue

Many mutations that impact protein function occur at residues that do not directly contact ligand. To understand the functional contributions from the sequence that links the DNA-binding and regulatory domains of the LacI/GalR homologues, we have created a chimeric protein (LLhP), which comprises the LacI DNA-binding domain, the LacI linker, and the PurR regulatory domain. Although DNA binding site residues are identical in LLhP and LacI, thermodynamic measurements of DNA binding affinity show that LLhP does not discriminate between alternative DNA ligands as well as LacI. In addition, small-angle scattering experiments show that LLhP is more compact than LacI. When DNA is released, LacI shows a 20 A increase in length that was previously attributed to unfolding of the linker. This change is not seen in apo-LLhP, even though the linker sequences of the two proteins are identical. Together, results indicate that long-range functional and structural changes are propagated across the interface that forms between the linker and regulatory domain. These changes could be mediated via the side chains of several linker residues that contact the regulatory domains of the naturally occurring proteins, LacI and PurR. Substitution of these residues in LLhP leads to a range of functional effects. Four variants exhibit altered affinity for DNA, with no changes in selectivity or allosteric response. Another two result in proteins that bind operator DNA with very low affinity and no allosteric response, similar to LacI binding nonspecific DNA sequences. Two more substitutions simultaneously diminish affinity, enhance allostery, and profoundly alter DNA ligand selectivity. Thus, positions within the linker can be varied to modulate different aspects of repressor function.     

Antibody discovery ex vivo accelerated by the LacO/LacI regulatory network

Monoclonal antibodies (mAbs) can be potent and highly specific therapeutics, diagnostics and research reagents. Nonetheless, mAb discovery using current in vivo or in vitro approaches can be costly and time-consuming, with no guarantee of success. We have established a platform for rapid discovery and optimization of mAbs ex vivo. This DTLacO platform derives from a chicken B cell line that has been engineered to enable rapid selection and seamless maturation of high affinity mAbs. We have validated the DTLacO platform by generation of high affinity and specific mAbs to five cell surface targets, the receptor tyrosine kinases VEGFR2 and TIE2, the glycoprotein TROP2, the small TNF receptor family member FN14, and the G protein-coupled receptor FZD10. mAb discovery is rapid and humanization is straightforward, establishing the utility of the DTLacO platform for identification of mAbs for therapeutic and other applications.     

Understand the Functions of Scaffold Proteins in Cell Signaling by a Mesoscopic Simulation Method

Scaffold proteins are central players in regulating the spatial-temporal organization of many important signaling pathways in cells. They offer physical platforms to downstream signaling proteins so that their transient interactions in a crowded and heterogeneous environment of cytosol can be greatly facilitated. However, most scaffold proteins tend to simultaneously bind more than one signaling molecule, which leads to the spatial assembly of multimeric protein complexes. The kinetics of these protein oligomerizations are difficult to quantify by traditional experimental approaches. To understand the functions of scaffold proteins in cell signaling, we developed a, to our knowledge, new hybrid simulation algorithm in which both spatial organization and binding kinetics of proteins were implemented. We applied this new technique to a simple network system that contains three molecules. One molecule in the network is a scaffold protein, whereas the other two are its binding targets in the downstream signaling pathway. Each of the three molecules in the system contains two binding motifs that can interact with each other and are connected by a flexible linker. By applying the new simulation method to the model, we show that the scaffold proteins will promote not only thermodynamics but also kinetics of cell signaling given the premise that the interaction between the two signaling molecules is transient. Moreover, by changing the flexibility of the linker between two binding motifs, our results suggest that the conformational fluctuations in a scaffold protein play a positive role in recruiting downstream signaling molecules. In summary, this study showcases the capability of computational simulation in understanding the general principles of scaffold protein functions.     

Utilization of Host Iron Sources by Corynebacterium diphtheriae: Multiple Hemoglobin-Binding Proteins Are Essential for the Use of Iron from the Hemoglobin-Haptoglobin Complex

The use of hemin iron by Corynebacterium diphtheriae requires the DtxR- and iron-regulated ABC hemin transporter HmuTUV and the secreted Hb-binding protein HtaA. We recently described two surface anchored proteins, ChtA and ChtC, which also bind hemin and Hb. ChtA and ChtC share structural similarities to HtaA; however, a function for ChtA and ChtC was not determined. In this study, we identified additional host iron sources that are utilized by C. diphtheriae. We show that several C. diphtheriae strains use the hemoglobin-haptoglobin (Hb-Hp) complex as an iron source. We report that an htaA deletion mutant of C. diphtheriae strain 1737 is unable to use the Hb-Hp complex as an iron source, and we further demonstrate that a chtA-chtC double mutant is also unable to use Hb-Hp iron. Single-deletion mutants of chtA or chtC use Hb-Hp iron in a manner similar to that of the wild type. These findings suggest that both HtaA and either ChtA or ChtC are essential for the use of Hb-Hp iron. Enzyme-linked immunosorbent assay (ELISA) studies show that HtaA binds the Hb-Hp complex, and the substitution of a conserved tyrosine (Y361) for alanine in HtaA results in significantly reduced binding. C. diphtheriae was also able to use human serum albumin (HSA) and myoglobin (Mb) but not hemopexin as iron sources. These studies identify a biological function for the ChtA and ChtC proteins and demonstrate that the use of the Hb-Hp complex as an iron source by C. diphtheriae requires multiple iron-regulated surface components.     

Oxidatively Damaged Erythrocytes Are Recognized by Membrane Proteins of Macrophages

Human erythrocytes incubated with an iron catalyst ADP-chelated Fe³⁺ undergo oxidative damage of the membrane including lipid peroxidation, protein oxidation, and protein aggregation, and become susceptible to recognition by human macrophages. In order to clarify the membrane components of macrophages responsible for the recogrution of the oxidized erythrocytes, binding of the oxidized cells to dot and Western blots of solubilized membrane of macrophages was investigated. The oxidized erythrocytes but not unoxidized cells bound to the dot blots. The binding was effectively inhibited by saccharide chains of band 3, a major glycoprotein of human erythrocytes, and lowered when the saccharide chains of band 3 were removed from the cell surface by pretreatment of the cells with endo-P-galactosidase which specifically cleaves the polylactosaminyl saccharide chains of band 3. The oxidized erythrocytes bound to the membrane proteins of macrophages with molecular mass of about 50, 80, and 120 kDa on Western blots depending on the saccharide chains of band 3 on their surface. The results suggest that the oxidatively damaged erythrocytes are specifically recognized by these proteins of macrophage membrane having saccharide binding ability.