András Láng

Structural Insights into the Trp‐Cage Folding Intermediate Formation

The 20 residue long Trp-cage is the smallest protein known, and thus has been the subject of several in vitro and in silico folding studies. Here, we report the multistate folding scenario of the miniprotein in atomic detail. We detected and characterized different intermediate states by temperature dependent NMR measurements of the (15)N and (13)C/(15)N labeled protein, both at neutral and acidic pH values. We developed a deconvolution technique to characterize the invisible--fully folded, unfolded and intermediate--fast exchanging states. Using nonlinear fitting methods we can obtain both the thermodynamic parameters (ΔH(F-I), T(m)(F-I), ΔC(p)(F-I) and ΔH(I-U), T(m)(I-U), ΔC(p)(I-U)) and the NMR chemical shifts of the conformers of the multistate unfolding process. During the unfolding of Trp-cage distinct intermediates evolve: a fast-exchanging intermediate is present under neutral conditions, whereas a slow-exchanging intermediate-pair emerges at acidic pH. The fast-exchanging intermediate has a native-like structure with a short α-helix in the G(11)-G(15) segment, whereas the slow-exchanging intermediate-pair presents elevated dynamics, with no detectable native-like residue contacts in which the G(11)-P(12) peptide bond has either cis or trans conformation. Heteronuclear relaxation studies combined with MD simulations revealed the source of backbone mobility and the nature of structural rearrangements during these transitions. The ability to detect structural and dynamic information about folding intermediates in vitro provides an excellent opportunity to gain new insights into the energetic aspects of the energy landscape of protein folding. Our new experimental data offer exceptional testing ground for further computational simulations.

Peptide models XLV: Conformational properties of N‐formyl‐L‐methioninamide and its relevance to methionine in proteins

The conformational space of the most biologically significant backbone folds of a suitable methionine peptide model was explored by density functional computational method. Using a medium [6‐31G(d)] and a larger basis set [6‐311++G(2d,2p)], the systematic exploration of low‐energy backbone structures restricted for the “L‐region” in the Ramachandran map of N‐formyl‐L‐methioninamide results in conformers corresponding to the building units of an extended backbone structure (βL), an inverse γ‐turn (γL), or a right‐handed helical structure (αL). However, no poly‐proline II type (ϵL) fold was found, indicating that this conformer has no intrinsic stability, and highlighting the effect of molecular environment in stabilizing this backbone structure. This is in agreement with the abundance of the ϵL‐type backbone conformation of methionine found in proteins. Stability properties (ΔE) and distinct backbone–side‐chain interactions support the idea that specific intramolecular contacts are operative in the “selection” of the lowest energy conformers. Apart from the number of different folds, all stable conformers are within a 10 kcal.mol−1 energy range, indicating the highly flexible behavior of methionine. This conformational feature can be important in supporting catalytic processes, facilitating protein folding and dimerization via metal ion binding. In both of the biological examples discussed (HIV‐1 reverse transcriptase and PcoC copper‐resistant protein), the conformational properties of Met residues were found to be of key importance. Spatial proximity to other types of residues or the same type of residue seems to be crucial for the structural integrity of a protein, whether Met is buried or exposed. Proteins 2005.

Dynll2 Dynein Light Chain Binds to an Extended Linear Motif of Myosin 5A Tail that Has Structural Plasticity.

LC8 dynein light chains (DYNLL) are conserved homodimeric eukaryotic hub proteins that participate in diverse cellular processes. Among the binding partners of DYNLL2, myosin 5a (myo5a) is a motor protein involved in cargo transport. Here we provide a profound characterization of the DYNLL2 binding motif of myo5a in free and DYNLL2-bound form by using nuclear magnetic resonance spectroscopy, X-ray crystallography, and molecular dynamics simulations. In the free form, the DYNLL2 binding region, located in an intrinsically disordered domain of the myo5a tail, has a nascent helical character. The motif becomes structured and folds into a β-strand upon binding to DYNLL2. Despite differences of the myo5a sequence from the consensus binding motif, one peptide is accommodated in each of the parallel DYNLL2 binding grooves, as for all other known partners. Interestingly, while the core motif shows a similar interaction pattern in the binding groove as seen in other complexes, the flanking residues make several additional contacts, thereby lengthening the binding motif. The N-terminal extension folds back and partially blocks the free edge of the β-sheet formed by the binding motif itself. The C-terminal extension contacts the dimer interface and interacts with symmetry-related residues of the second myo5a peptide. The involvement of flanking residues of the core binding site of myo5a could modify the quaternary structure of the full-length myo5a and affect its biological functions. Our results deepen the knowledge of the diverse partner recognition of DYNLL proteins and provide an example of a Janus-faced linear motif.

Development of Cyclic NGR Peptides with Thioether Linkage: Structure and Dynamics Determining Deamidation and Bioactivity

NGR peptides that recognize CD13 receptors in tumor neovasculature are of high interest, in particular due to their potential applications in drug targeting. Here we report the synthesis and structural analysis of novel thioether bond-linked cyclic NGR peptides. Our results show that their chemostability (resistance against spontaneous decomposition forming isoAsp and Asp derivatives) strongly depends on both sample handling conditions and structural properties. A significant correlation was found between chemostability and structural measures, such as NH(Gly)-CO(Asn-sc) distances. The side-chain orientation of Asn is a key determining factor; if it is turned away from HN(Gly), the chemostability increases. Structure stabilizing factors (e.g., H-bonds) lower their internal dynamics, and thus biomolecules become even more resistant against spontaneous decomposition. The effect of cyclic NGR peptides on cell adhesion was examined in A2058 melanoma cell lines. It was found that some of the investigated peptides gradually increased cell adhesion with long-term characteristics, indicating time-dependent formation of integrin binding isoAsp derivatives that are responsible for the adhesion-inducing effect.

Challenging drug target for Parkinson's disease: Pathological complex of the chameleon TPPP/p25 and alpha-synuclein proteins

The hallmarks of Parkinsons disease and other synucleinopathies, Tubulin Polymerization Promoting Protein (TPPP/p25) and α-synuclein (SYN) have two key features: they are disordered and co-enriched/co-localized in brain inclusions. These Neomorphic Moonlighting Proteins display both physiological and pathological functions due to their interactions with distinct partners. To achieve the selective targeting of the pathological TPPP/p25-SYN but not the physiological TPPP/p25-tubulin complex, their interfaces were identified as a specific innovative strategy for the development of anti-Parkinson drugs. Therefore, the interactions of TPPP/p25 with tubulin and SYN were characterized which suggested the involvements of the 178-187 aa and 147-156 aa segments in the complexation of TPPP/p25 with tubulin and SYN, respectively. However, various truncated and deletion mutants reduced but did not abolish the interactions except one mutant; in addition synthetized fragments corresponding to the potential binding segments of TPPP/p25 failed to interact with SYN. In fact, the studies of the multiple interactions at molecular and cellular levels revealed the high conformational plasticity, chameleon feature, of TPPP/p25 that ensures exceptional functional resilience; the lack of previously identified binding segments could be replaced by other segments. The experimental results are underlined by distinct bioinformatics tools. All these data revealed that although targeting chameleon proteins is a challenging task, nevertheless, the validation of a drug target can be achieved by identifying the interface of complexes of the partner proteins existing at the given pathological conditions.

Intermodule cooperativity in the structure and dynamics of consecutive complement control modules in human C1r

The modular C1r protein is the first protease activated in the classical complement pathway, a key component of innate immunity. Activation of the heteropentameric C1 complex, possibly accompanied by major intersubunit re‐arrangements besides proteolytic cleavage, requires targeted regulation of flexibility within the context of the intramolecular and intermolecular interaction networks of the complex. In this study, we prepared the two complement control protein (CCP) modules, CCP1 and CCP2, of C1r in their free form, as well as their tandem‐linked construct, CCP1CCP2, to characterize their solution structure, conformational dynamics and cooperativity. The structures derived from NMR signal dispersion and secondary chemical shifts were in good agreement with those obtained by X‐ray crystallography. However, successful heterologus expression of both the single CCP1 module and the CCP1CCP2 constructs required the attachment of the preceding N‐terminal module, CUB2, which could then be removed to obtain the properly folded proteins. Internal mobility of the modules, especially that of CCP1, exhibited considerable changes accompanied by interfacial chemical shift alterations upon the attachment of the C‐terminal CCP2 domain. Our NMR data suggest that in terms of folding, stability and dynamics, CCP1 is heavily dependent on the presence of its neighboring modules in intact C1r. Therefore, CCP1 could be a focal interaction point, capable of transmitting information towards its neighboring modules.

Multiple fuzzy interactions in the moonlighting function of thymosin-β4

Thymosine β4 (Tß4) is a 43 amino acid long intrinsically disordered protein (IDP), which was initially identified as an actin-binding and sequestering molecule. Later it was described to have multiple other functions, such as regulation of endothelial cell differentiation, blood vessel formation, wound repair, cardiac cell migration, and survival.1 The various functions of Tβ4 are mediated by interactions with distinct and structurally unrelated partners, such as PINCH, ILK, and stabilin-2, besides the originally identified G-actin. Although the cellular readout of these interactions and the formation of these complexes have been thoroughly described, no attempt was made to study these interactions in detail, and to elucidate the thermodynamic, kinetic, and structural underpinning of this range of moonlighting functions. Because Tβ4 is mostly disordered, and its 4 described partners are structurally unrelated (the CTD of stabilin-2 is actually fully disordered), it occurred to us that this system might be ideal to characterize the structural adaptability and ensuing moonlighting functions of IDPs. Unexpectedly, we found that Tβ4 engages in multiple weak, transient, and fuzzy interactions, i.e., it is capable of mediating distinct yet specific interactions without adapting stable folded structures.

Interaction between separated consecutive complement control modules of human C1r: Implications for dimerization of the full-length protease

MINT‐8045767: CCP1 (uniprotkb:P00736) and CCP2 (uniprotkb:P00736) bind (MI:0407) by nuclear magnetic resonance (MI:0077)