Electrostatic Optimization of the Conformational Energy Landscape in a Metamorphic Protein

The equilibrium unfolding reaction of Ltn, a metamorphic C-class chemokine, was monitored by tryptophan fluorescence to determine unfolding free energies. Measurements revealed that addition of 150 mM NaCl stabilized the Ltn chemokine fold by approximately 1 kcal/mol. Specific mutations involving Arg23 and Arg43 also increased the stability by 1 kcal/mol, suggesting their involvement in chloride ion coordination. This interaction was confirmed by nuclear magnetic resonance (NMR) salt titration studies that revealed chemical shift perturbations localized to these residues and backbone amides within the proximal 40s loop. The effects of NaCl on the free energy landscape were further verified by ZZ-exchange NMR spectroscopy. Our results suggest that changes in the electrostatic environment modulate the Gibbs free energy of folding and alter the forward and reverse rates of interconversion. These results demonstrate how solution ions can promote metamorphic folding by adjusting the relative stabilities of two unrelated Ltn native-state structures

Allosteric Activation of the Par‑6 PDZ via a Partial Unfolding Transition

Proteins exist in a delicate balance between the native and unfolded states, where thermodynamic stability may be sacrificed to attain the flexibility required for efficient catalysis, binding, or allosteric control. Partition-defective 6 (Par-6) regulates the Par polarity complex by transmitting a GTPase signal through the Cdc42/Rac interaction binding PSD-95/Dlg/ZO-1 (CRIB-PDZ) module that alters PDZ ligand binding. Allosteric activation of the PDZ is achieved by local rearrangement of the L164 and K165 side chains to stabilize the interdomain CRIB:PDZ interface and reposition a conserved element of the ligand binding pocket. However, microsecond to millisecond dynamics measurements revealed that L164/K165 exchange requires a larger rearrangement than expected. The margin of thermodynamic stability for the PDZ domain is modest (∼3 kcal/mol) and further reduced by transient interactions with the disordered CRIB domain. Measurements of local structural stability revealed that tertiary contacts within the PDZ are disrupted by a partial unfolding transition that enables interconversion of the L/K switch. The unexpected participation of partial PDZ unfolding in the allosteric mechanism of Par-6 suggests that native-state unfolding may be essential for the function of other marginally stable proteins

Engineering Metamorphic Chemokine Lymphotactin/XCL1 into the GAG-Binding, HIV-Inhibitory Dimer Conformation

Unlike other chemokines, XCL1 undergoes a distinct metamorphic interconversion between a canonical monomeric chemokine fold and a unique β-sandwich dimer. The monomeric conformation binds and activates the receptor XCR1, whereas the dimer binds extracellular matrix glycosaminoglycans and has been associated with anti-human immunodeficiency virus (HIV) activity. Functional studies of WT-XCL1 are complex, as both conformations are populated in solution. To overcome this limitation, we engineered a stabilized dimeric variant of XCL1 designated CC5. This variant features a new disulfide bond (A36C–A49C) that prevents structural interconversion by locking the chemokine into the β-sandwich dimeric conformation, as demonstrated by NMR structural analysis and hydrogen/deuterium exchange experiments. Functional studies analyzing glycosaminoglycan binding demonstrate that CC5 binds with high affinity to heparin. In addition, CC5 exhibits potent inhibition of HIV-1 activity in primary peripheral blood mononuclear cells (PBMCs), demonstrating the importance of the dimer in blocking viral infection. Conformational variants like CC5 are valuable tools for elucidating the biological relevance of the XCL1 native-state interconversion and will assist in future antiviral and functional studies

Binding of Crumbs to the Par‑6 CRIB-PDZ Module Is Regulated by Cdc42

Par-6 is a scaffold protein that organizes other proteins into a complex required to initiate and maintain cell polarity. Cdc42-GTP binds the CRIB module of Par-6 and alters the binding affinity of the adjoining PDZ domain. Allosteric regulation of the Par-6 PDZ domain was first demonstrated using a peptide identified in a screen of typical carboxyl-terminal ligands. Crumbs, a membrane protein that localizes a conserved polarity complex, was subsequently identified as a functional partner for Par-6 that likely interacts with the PDZ domain. Here we show by nuclear magnetic resonance that Par-6 binds a Crumbs carboxyl-terminal peptide and report the crystal structure of the PDZ–peptide complex. The Crumbs peptide binds Par-6 more tightly than the previously studied carboxyl peptide ligand and interacts with the CRIB-PDZ module in a Cdc42-dependent manner. The Crumbs:Par-6 crystal structure reveals specific PDZ–peptide contacts that contribute to its higher affinity and Cdc42-enhanced binding. Comparisons with existing structures suggest that multiple C-terminal Par-6 ligands respond to a common conformational switch that transmits the allosteric effects of GTPase binding

The Aspartate-Less Receiver (ALR) Domains: Distribution, Structure and Function

<div><p>Two-component signaling systems are ubiquitous in bacteria, Archaea and plants and play important roles in sensing and responding to environmental stimuli. To propagate a signaling response the typical system employs a sensory histidine kinase that phosphorylates a Receiver (REC) domain on a conserved aspartate (Asp) residue. Although it is known that some REC domains are missing this Asp residue, it remains unclear as to how many of these divergent REC domains exist, what their functional roles are and how they are regulated in the absence of the conserved Asp. Here we have compiled all deposited REC domains missing their phosphorylatable Asp residue, renamed here as the Aspartate-Less Receiver (ALR) domains. Our data show that ALRs are surprisingly common and are enriched for when attached to more rare effector outputs. Analysis of our informatics and the available ALR atomic structures, combined with structural, biochemical and genetic data of the ALR archetype RitR from <i>Streptococcus pneumoniae</i> presented here suggest that ALRs have reorganized their active pockets to instead take on a constitutive regulatory role or accommodate input signals other than Asp phosphorylation, while largely retaining the canonical post-phosphorylation mechanisms and dimeric interface. This work defines ALRs as an atypical REC subclass and provides insights into shared mechanisms of activation between ALR and REC domains.</p></div

Structural Analysis of a Novel Small Molecule Ligand Bound to the CXCL12 Chemokine

CXCL12 binds to CXCR4, promoting both chemotaxis of lymphocytes and metastasis of cancer cells. We previously identified small molecule ligands that bind CXCL12 and block CXCR4-mediated chemotaxis. We now report a 1.9 Å resolution X-ray structure of CXCL12 bound by such a molecule at a site normally bound by sY21 of CXCR4. The complex structure reveals binding hot spots for future inhibitor design and suggests a new approach to targeting CXCL12–CXCR4 signaling in drug discovery

Crystal structure of the RitR REC domain.

<p>(<b>a</b>) Cartoon representation of RitR_ALR with helices α1- α3 and α5 colored orange, the unusual α4 helix colored green, and the β-strands colored blue. The equivalent of the phospho-modified Asp residue found in typical REC domains, RitR coordinate Asn53, is shown as ball-and-stick with blue carbon and red oxygen atoms. (<b>b</b>) Schematic view showing the pattern of RitR van der Waals interactions (yellow dotted lines) and hydrogen-bonding network (green dotted lines) in the dimer / Gate region of the structure. (<b>c</b>) Close-up of the kinked α4 helix (in green) and surrounding residues. The blue sphere is a water molecule.</p

ALR statistics and phylogeny.

<p>(<b>a</b>) Frequency of amino acid substitutions within six key ‘invariant’ REC residues in ALR sequences: the (now changed in ALRs) conserved histidine kinase phosphorylated aspartate residue position that defines the ALR subfamily (Phospho-Asp), acidic triad residue-1 (Glu9 in RitR) and acid triad residue-2 (Lys10 in RitR) that normally help coordinate the metal ion active pocket, the Tyrosine/Phenylalanine (Tyr/Phe, Tyr100 in RitR) and Threonine/Serine (Thr/Ser, Asp81 in RitR) that make up the Y/T-coupling system, and the conserved pocket Lys (Lys103 in RitR). Notice that where catalytic active pocket Asp/Lys residues have often been changed in ALR sequences (top panel), the T/Y-coupling residues generally remain conserved (bottom panel). This trend in conservation is also observed for the acidic triad-1 and the universally conserved pocket Lys residue (Lys103 in RitR), but not for acidic triad-2. (<b>b</b>) Taxonomic distribution of ALR sequences. The number of ALRs discovered in the given class or phylum is shown in parentheses. (<b>c</b>) Distribution of the average number of ALR sequences per completed genome by phyla. (<b>d</b>) Bar graph of the percentage contribution of a given Effector Domain (ED) within total REC sequences (shown as black bars) and ALR sequences only (shown as non-black bars). An asterisk above the bars indicates that ALRs are enriched for the ED by over 50% within the ALR population compared to their representation within typical REC sequence populations. An asterisk in front of the ED name indicates that the ALR or REC domain is (unusually) C-terminal to the ED sequence.</p

Crystallographic data collection and model refinement statistics.

<p>^aValues in parentheses apply to the high-resolution shell indicated in the resolution row.</p><p>^bR = Σ(||Fobs|-scale*|Fcalc||) / Σ |Fobs|.</p><p>^cNumber of close interatomic contacts per 1000 atoms.</p><p>^dIsotropic equivalent B factors, including contribution from TLS refinement.</p><p>Crystallographic data collection and model refinement statistics.</p

Structure of the RitR ‘active’ pocket.

<p>(<b>a</b>) Stereoview of the electron density in the RitR active site (magenta mesh) contoured at 1.5 σ. Water molecules are shown as blue spheres. Notice the lack of a metal ion in the typical metal-binding site near Glu9. (<b>b</b>) Schematic view of the RitR REC ‘active site’ showing predicted hydrogen-bonding interactions (green dotted lines) with distances in Angstroms (Å). (<b>c</b>) Comparison of the vacuum electrostatic surface potentials of RitR_ALR, <i>left</i>, and the PhoB REC domain, <i>right</i>. The Mg²⁺ site in PhoB is indicated by a magenta sphere, which can be seen protruding slightly through the surface (denoted by the white arrow). (<b>d</b>) Comparison of the surfaces of the RitR and PhoB REC domains colored by distance from the center of mass of each protein. Not only is the electronegative environment in the metal-binding site of PhoB lost in RitR, the cleft that normally holds the metal ion (yellow region near the white arrow) is missing as well. Figure generated using PyMol (Version 1.4.1, Schrödinger, LLC).</p