Protein-protein interactions mediated by modular protein domains are critical for cell scaffolding, differentiation, signaling, and ultimately, evolution. Given the vast number of ligands competing for binding to a limited number of domain families, it is often puzzling how specificity can be achieved. Selectivity may be modulated by intradomain allostery, whereby a remote residue is energetically connected to the functional binding site via side chain or backbone interactions. Whereas several energetic pathways, which could mediate intradomain allostery, have been predicted in modular protein domains, there is a paucity of experimental data to validate their existence and roles. Here, we have identified such functional energetic networks in one of the most common protein-protein interaction modules, the PDZ domain. We used double mutant cycles involving site-directed mutagenesis of both the PDZ domain and the peptide ligand, in conjunction with kinetics to capture the fine energetic details of the networks involved in peptide recognition. We performed the analysis on two homologous PDZ-ligand complexes and found that the energetically coupled residues differ for these two complexes. This result demonstrates that amino acid sequence rather than topology dictates the allosteric pathways. Furthermore, our data support a mechanism whereby the whole domain and not only the binding pocket is optimized for a specific ligand. Such cross-talk between binding sites and remote residues may be used to fine tune target selectivity.Several studies on protein-protein interaction modules (1), such as SH2, SH3, and PDZ 5 domains, have highlighted the apparent problem of how selectivity is achieved in a cellular context (2-4). For example, the synapse contains several PDZ proteins (5) with apparently overlapping specificity, and yet they have distinct roles in scaffolding and signaling (6). One mechanism that could modulate specificity is intradomain allostery (7). Multisubunit proteins such as hemoglobin are well known for their allosteric behavior whereby the oxygen binding is cooperative because ligation of one subunit will modulate the affinity in the other subunits of the tetramer (8). But allostery is also present within monomeric proteins (9), and it may be invoked even in the absence of a detectable conformational change (10 -15).Previous studies on monomeric globular proteins have predicted allosteric pathways on an amino acid residue level based on analysis of co-evolution (16, 17), molecular dynamics simulations (18), or NMR-constrained molecular dynamics (19). Although the PDZ domain family has served as a prime model system for these studies, experimental data that detect intramolecular allosteric pathways are scarce and sometimes conflicting (16,20,21).In protein folding studies, it proved illuminating to compare homologous proteins to unravel basic determinants of the underlying mechanism (22). Here, we employ the same strategy to assess the structural pattern of intradomain allostery by looking at the coupling fr...

Human transferrin receptor 1 (CD71) guarantees iron supply by endocytosis upon binding of iron-loaded transferrin and ferritin. Arenaviruses and the malaria parasite exploit CD71 for cell invasion and epitopes on CD71 for interaction with transferrin and pathogenic hosts were identified. Here, we provide the molecular basis of the CD71 ectodomain-human ferritin interaction by determining the 3.9 Å resolution single-particle cryo-electron microscopy structure of their complex and by validating our structural findings in a cellular context. The contact surfaces between the heavy-chain ferritin and CD71 largely overlap with arenaviruses and Plasmodium vivax binding regions in the apical part of the receptor ectodomain. Our data account for transferrin-independent binding of ferritin to CD71 and suggest that select pathogens may have adapted to enter cells by mimicking the ferritin access gate.

Intrinsically disordered proteins are very common and mediate numerous protein-protein and protein-DNA interactions. While it is clear that these interactions are instrumental for the life of the mammalian cell, there is a paucity of data regarding their molecular binding mechanisms. We have here used short peptides as a model system for intrinsically disordered proteins. Linear free-energy relationships based on rate and equilibrium constants for the binding of these peptides to ordered target proteins, PDZ domains, demonstrate that native side-chain interactions form mainly after the ratelimiting barrier for binding, in a cooperative fashion. This finding suggests that these disordered peptides first form a weak encounter complex with non-native interactions.The data do not support the recent notion that the affinities of intrinsically disordered proteins towards their targets are generally governed by their association rate constants.Instead, we observe the opposite for peptide-PDZ interactions, namely that changes in K d correlate with changes in k off .3

The regulation of cytochrome P450 activity is often achieved by structural transitions induced by substrate binding. We describe the conformational transition experienced upon binding by the P450 OleP, an epoxygenase involved in oleandomycin biosynthesis. OleP bound to the substrate analog 6DEB crystallized in 2 forms: one with an ensemble of open and closed conformations in the asymmetric unit and another with only the closed conformation. Characterization of OleP-6DEB binding kinetics, also using the P450 inhibitor clotrimazole, unveiled a complex binding mechanism that involves slow conformational rearrangement with the accumulation of a spectroscopically detectable intermediate where 6DEB is bound to open OleP. Data reported herein provide structural snapshots of key precatalytic steps in the OleP reaction and explain how structural rearrangements induced by substrate binding regulate activity.-Parisi, G., Montemiglio, L. C., Giuffrè, A., Macone, A., Scaglione, A., Cerutti, G., Exertier, C., Savino, C., Vallone, B. Substrate-induced conformational change in cytochrome P450 OleP.

Neuroglobin (Ngb) is predominantly expressed in neurons of the central and peripheral nervous systems and it clearly seems to be involved in neuroprotection. Engineering Ngb to observe structural and dynamic alterations associated with perturbation in ligand binding might reveal important structural determinants, and could shed light on key features related to its mechanism of action. Our results highlight the relevance of the CD loop and of Phe106 as distal and proximal controls involved in ligand binding in murine neuroglobin. We observed the effects of individual and combined mutations of the CD loop and Phe106 that conferred to Ngb higher CO binding velocities, which we correlate with the following structural observations: the mutant F106A shows, upon CO binding, a reduced heme sliding hindrance, with the heme present in a peculiar double conformation, whereas in the CD loop mutant “Gly-loop”, the original network of interactions between the loop and the heme was abolished, enhancing binding via facilitated gating out of the distal His64. Finally, the double mutant, combining both mutations, showed a synergistic effect on CO binding rates. Resonance Raman spectroscopy and MD simulations support our findings on structural dynamics and heme interactions in wild type and mutated Ngbs.

Neuroglobin is a member of the globin family involved in neuroprotection; it is primarily expressed in the brain and retina of vertebrates. Neuroglobin belongs to the heterogeneous group of hexacoordinate globins that have evolved in animals, plants and bacteria, endowed with the capability of reversible intramolecular coordination, allowing the binding of small gaseous ligands (O2, NO and CO). In a unique fashion among haemoproteins, ligand-binding events in neuroglobin are dependent on the sliding of the haem itself within a preformed internal cavity, as revealed by the crystal structure of its CO-bound derivative. Point mutants of the neuroglobin internal cavity have been engineered and their functional and structural characterization shows that hindering the haem displacement leads to a decrease in CO affinity, whereas reducing the cavity volume without interfering with haem sliding has negligible functional effects.

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Different murine neuroglobin variants showing structural and dynamic alterations that are associated with perturbation of ligand binding have been studied: the CD loop mutants characterized by an enhanced flexibility (Gly‐loop40–48 and Gly‐loop44–47), the F106A mutant, and the double Gly‐loop44–47/F106A mutant. Their ferric resonance Raman spectra in solution and in crystals are almost identical. In the high‐frequency region, the identification of a double set of core size marker bands indicates the presence of two 6‐coordinate low spin species. The resonance Raman data, together with the corresponding crystal structures, indicate the presence of two neuroglobin conformers with a reversed (A conformer) or a canonical (B conformer) heme insertion orientation. With the identification of the marker bands corresponding to each conformer, the data indicate that the B conformer increases at the expense of the A form, predominantly in the Gly‐loop44–47/F106A double mutant, as confirmed by X‐ray crystallography. This is the first time that a reversed heme insertion has been identified by resonance Raman in a native 6‐coordinate low‐spin heme protein. This diagnostic tool could be extended to other heme proteins in order to detect heme orientational disorder, which are likely to be correlated to functionally relevant heme dynamics. Database Crystallographic structure: structural data are deposited in the Protein Data Bank under the http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6RA6 PDB entry.