Magnesium Activates Microsecond Dynamics to Regulate Integrin-Collagen Recognition.

Integrin receptors bind collagen via metal-mediated interactions that are modulated by magnesium (Mg2+) levels in the extracellular matrix. Nuclear magnetic resonance-based relaxation experiments, isothermal titration calorimetry, and adhesion assays reveal that Mg2+ functions as both a structural anchor and dynamic switch of the α1ß1 integrin I domain (α1I). Specifically, Mg2+ binding activates micro- to millisecond timescale motions of residues distal to the binding site, particularly those surrounding the salt bridge at helix 7 and near the metal ion-dependent adhesion site. Mutagenesis of these residues impacts α1I functional activity, thereby suggesting that Mg-bound α1I dynamics are important for collagen binding and consequent allosteric rearrangement of the low-affinity closed to high-affinity open conformation. We propose a multistep recognition mechanism for α1I-Mg-collagen interactions involving both conformational selection and induced-fit processes. Our findings unravel the multifaceted role of Mg2+ in integrin-collagen recognition and assist in elucidating the molecular mechanisms by which metals regulate protein-protein interactions.

Revealing Accessibility of Cryptic Protein Binding Sites within the Functional Collagen Fibril.

Fibrillar collagens are the most abundant proteins in the extracellular matrix. Not only do they provide structural integrity to all of the connective tissues in the human body, but also their interactions with multiple cell receptors and other matrix molecules are essential to cell functions, such as growth, repair, and cell adhesion. Although specific binding sequences of several receptors have been determined along the collagen monomer, processes by which collagen binding partners recognize their binding sites in the collagen fibril, and the critical driving interactions, are poorly understood. The complex molecular assembly of bundled triple helices within the collagen fibril makes essential ligand binding sites cryptic or hidden from the molecular surface. Yet, critical biological processes that require collagen ligands to have access to interaction sites still occur. In this contribution, we will discuss the molecular packing of the collagen I fibril from the perspective of how collagen ligands access their known binding regions within the fibril, and we will present our analysis of binding site accessibility from the fibril surface. Understanding the basis of these interactions at the atomic level sets the stage for developing drug targets against debilitating collagen diseases and using collagen as drug delivery systems and new biomaterials.

Intrinsic local destabilization of the C-terminus predisposes integrin α1 I domain to a conformational switch induced by collagen binding.

Integrin-collagen interactions play a critical role in a myriad of cellular functions that include immune response, and cell development and differentiation, yet their mechanism of binding is poorly understood. There is increasing evidence that conformational flexibility assumes a central role in the molecular mechanisms of protein-protein interactions and here we employ NMR hydrogen-deuterium exchange (HDX) experiments to explore the impact of slower timescale dynamic events. To gain insight into the mechanisms underlying collagen-induced conformational switches, we have undertaken a comparative study between the wild type integrin α1 I and a gain-of-function E317A mutant. NMR HDX results suggest a relationship between regions exhibiting a reduced local stability in the unbound I domain and those that undergo significant conformational changes upon binding. Specifically, the αC and α7 helices within the C-terminus are at the center of such major perturbations and present reduced local stabilities in the unbound state relative to other structural elements. Complementary isothermal titration calorimetry experiments have been performed to derive complete thermodynamic binding profiles for association of the collagen-like triple-helical peptide with wild type α1 I and E317A mutant. The differential energetics observed for E317A are consistent with the HDX experiments and support a model in which intrinsically destabilized regions predispose conformational rearrangement in the integrin I domain. This study highlights the importance of exploring different timescales to delineate allosteric and binding events.

A peptide study of the relationship between the collagen triple-helix and amyloid.

Type XXV collagen, or collagen-like amyloidogenic component, is a component of amyloid plaques, and recent studies suggest this collagen affects amyloid fibril elongation and has a genetic association with Alzheimer's disease. The relationship between the collagen triple helix and amyloid fibrils was investigated by studying peptide models, including a very stable triple helical peptide (Pro-Hyp-Gly)10 , an amyloidogenic peptide GNNQQNY, and a hybrid peptide where the GNNQQNY sequence was incorporated between (GPO)(n) domains. Circular dichroism and nuclear magnetic resonance (NMR) spectroscopy showed the GNNQQNY peptide formed a random coil structure, whereas the hybrid peptide contained a central disordered GNNQQNY region transitioning to triple-helical ends. Light scattering confirmed the GNNQQNY peptide had a high propensity to form amyloid fibrils, whereas amyloidogenesis was delayed in the hybrid peptide. NMR data suggested the triple-helix constraints on the GNNQQNY sequence within the hybrid peptide may disfavor the conformational change necessary for aggregation. Independent addition of a triple-helical peptide to the GNNQQNY peptide under aggregating conditions delayed nucleation and amyloid fibril growth. The inhibition of amyloid nucleation depended on the Gly-Xaa-Yaa sequence and required the triple-helix conformation. The inhibitory effect of the collagen triple-helix on an amyloidogenic sequence, when in the same molecule or when added separately, suggests Type XXV collagen, and possibly other collagens, may play a role in regulating amyloid fibril formation.

DNA strand breakage induced by CuII and NiII, in the presence of peptide models of histone H2B.

In the present study we used the plasmid relaxation assay, a very sensitive method for detection of DNA strand breaks in vitro, in order to evaluate the role of peptide fragments of histone H2B in DNA strand breakage induced by copper and nickel. We have found that in the presence of peptides modeling the histone fold domain (H2B(32-62) and H2B(63-93)) as well as the N-terminal tail (H2B(1-31)) of histone H2B there is an increased DNA damage by Cu(2+)/H(2)O(2) and Ni(2+)/H(2)O(2) reaction mixtures. On the contrary, the C-terminal tail (H2B(94-125)) seems to have a protective role on the attack of ROS species to DNA. We have rendered our findings to the interactions of the peptides with DNA, as well as with the metal.

Copper effective binding with 32-62 and 94-125 peptide fragments of histone H2B.

In an attempt to investigate the role of histone H2B in Cu(II) induced toxicity and carcinogenesis, we synthesized the terminally blocked peptides H2B(32-62) (SRKESYSVYVYKVLKQVH(48)PDTGISSKAMGIM) and Η2Β(94-125) (IQTAVRLLLPGELAKH(110)AVSEGTKAVTKYTSS), mimicking the N-terminal histone-fold domain and C-terminal tail of histone H2B, respectively and studied their interaction with Cu(II) ions by means of potentiometric titrations and spectroscopic techniques (UV-visible, CD and EPR). Both peptides, H2B(32-62) and H2B(94-125), interacted efficiently with Cu(II) ions, forming several species from pH 4 to 11, with His(48) and His(110) serving as anchors for metal binding. In H2B(32-62), the effective Cu(II) binding is emphasized by the formation of a soluble Cu(II)-H2B(32-62) complex, unlike the unbound peptide that precipitated over pH 7.9. At physiological pH, both peptides form tetragonal 3N species with a {N(Im), 2N(-)} coordination mode. At this pH, H2B(32-62) presented the formation of coordination isomers, differentiated by the presence in one of them, of an axial coordination of the carboxylate group of Asp(50). Copper binding with both H2B(32-62) and H2B(94-125) may induce a conformational change in the peptides' original structure. At physiological conditions, this effect may interfere with nucleosome's structure and dynamics, including the ubiquitination of Lys(120) which is linked to gene silencing.

The possible role of 94-125 peptide fragment of histone H2B in nickel-induced carcinogenesis.

The molecular mechanism by which nickel carcinogenicity is exerted is not fully understood. However, it is believed to involve DNA damage and epigenetic effects in chromatin, resulting from metal binding to the cell nucleus. Histone nuclear proteins are the major candidates for metal binding not only due to their abundance but also due to the presence of strong binding sites within their sequence. In order to investigate the binding capacity of histone H2B toward Ni(2+) ions, we synthesized the peptide Ac-IQTAVRLLLPGELAKHAVSEGTKAVTKYTSSK-Am (H2B(94-125)) as a model of the C-terminal tail. Complexation of H2B(94-125) with Ni(2+) starts at pH around 5 with the formation of a distorted octahedral complex. Over pH 8, this species shifts to a square-planar geometry, with the complete consumption of free Ni(2+) ions at pH 10. The formation of the diamagnetic square-planar complex was further studied by means of NMR spectroscopy. On the basis of the NOE connectivities we determined a well-resolved solution structure for the binding site of the H2B(94-125)-Ni(2+) complex, including residues E(12)LAKHAVS(19). Interestingly, nickel binding strongly affects the C-terminal of the peptide, forcing it to approach the coordination plane. If such a structural alteration is able to occur under physiological conditions, it is highly possible that it interferes with the histone's physiological role and particularly with the ubiquitination process, taking place at Lys(120). We believe that these findings will assist in a better understanding of the role of histone H2B in the mechanisms of metal-induced toxicity and carcinogenesis.

Coordination of Cu(2+)and Ni(2+) with the histone model peptide of H2B N-terminal tail (1-31 residues): A spectroscopic study.

The interaction of Cu(2+) and Ni(2+) with the N-terminal tail of histone H2B, the 31 amino acid peptide H2B(1-31) (Ac-PEPAKSAPAPKKG(13)SKKAVTKAQKKD(25)GKKRKR-NH(2)), studied by various spectroscopic techniques (UV/Vis, CD, EPR and NMR) are described. The results showed the formation of Cu(2+)-H2B(1-31) complexes above pH 7.3 most probably through the beta-carboxylate group of D25. With the increase of the pH, a mixture of 3 N and 4 N species presenting {2N(-), CO, epsilonNH(2)} and {2N(-), OH(2), epsilonNH(2)}{epsilonNH(2)} coordination modes, respectively is formed, while at highly basic solutions the binding of an additional amide donor is suggested. NMR spectroscopy supported by CD spectroscopy indicated that Ni(2+) coordination takes place most likely through Q22-K23-K24-D25 peptide fragment. Direct coordination to Ni(2+), in a {4N(-)} coordination mode, with a severe conformation change in all residues from G13 to G26 was observed. Cu(2+) and Ni(2+) binding to the N-terminal tail of H2B causes a severe conformational change that might interfere with histone post-translational modifications, possibly leading to epigenetic changes.

Interaction of histone H2B (fragment 63-93) with Ni(ii). An NMR study.

The behaviour of the 31 mer peptide (Ac-NSFVNDIFERIAG(13)EASRL(18)A(19)H(20)YNKRS(25)TITSRE-NH(2)), modelling the histone-fold domain (63 to 93 residues) of H2B, towards Ni(ii) was investigated by multidimensional NMR spectroscopy (1D, 2D TOCSY, NOESY and (13)C-HSQC). The coordination involved the imidazole of His20 and three amide nitrogens of His20, Ala19 and Leu18, similar to the one shown by the hexapeptide LAHYNK contained in the 31 mer peptide. The solution structure of the Ni(ii) complex with the tridecapeptide comprising histone's H2B 75-87 residues, was elucidated from the NOE cross correlations observed in the 2D-NOESY spectrum. A severe change in the peptide's conformation was observed, passing from a partially helical to a well-defined ordered structure around the metal ion. A remarkable structural feature is the position of the aromatic ring of Tyr21 below the coordination plane. This and the hydrophobic fence created by Leu18 and Ala19, together with the position of Arg17 and Arg24 side chains seem to be relevant to the complex stability. We believe that these structural modifications may be physiologically important in the mechanism of nickel induced carcinogenesis.

Interaction of Cu(II) and Ni(II) with the 63-93 fragment of histone H2B.

Chromatin proteins are believed to represent reactive sites for metal ion binding. We have synthesized the 31 amino acid peptide Ac-NSFVNDIFERIAGEASRLAHYNKRSTITSRE-NH2, corresponding to the 63-93 fragment of the histone H2B and studied its interaction with Cu(II) and Ni(II). Potentiometric and spectroscopic studies (UV-vis, CD, NMR and EPR) showed that histidine 21 acts as an anchoring binding site for the metal ion. Complexation of the studied peptide with Cu(II) starts at pH 4 with the formation of the monodentate species CuH2L. At physiological pH values, the 3N complex (N(Im), 2N(-)), CuL is favoured while at basic pH values the 4N (N(Im), 3N(-)) coordination mode is preferred. Ni(II) forms several complexes with the peptide starting from the distorted octahedral NiH2L at about neutral pH, to a square planar complex where the peptide is bound through a (N(Im), 3N(-)) mode in an equatorial plane at basic pH values. These results could be important in revealing more information about the mechanism of metal induced toxicity and carcinogenesis.