Guido Scarabelli

Predicting Interaction Sites from the Energetics of Isolated Proteins: A New Approach to Epitope Mapping

An increasing number of functional studies of proteins have shown that sequence and structural similarities alone may not be sufficient for reliable prediction of their interaction properties. This is particularly true for proteins recognizing specific antibodies, where the prediction of antibody-binding sites, called epitopes, has proven challenging. The antibody-binding properties of an antigen depend on its structure and related dynamics. Aiming to predict the antibody-binding regions of a protein, we investigate a new approach based on the integrated analysis of the dynamical and energetic properties of antigens, to identify nonoptimized, low-intensity energetic interaction networks in the protein structure isolated in solution. The method is based on the idea that recognition sites may correspond to localized regions with low-intensity energetic couplings with the rest of the protein, which allows them to undergo conformational changes, to be recognized by a binding partner, and to tolerate mutations with minimal energetic expense. Upon analyzing the results on isolated proteins and benchmarking against antibody complexes, it is found that the method successfully identifies binding sites located on the protein surface that are accessible to putative binding partners. The combination of dynamics and energetics can thus discriminate between epitopes and other substructures based only on physical properties. We discuss implications for vaccine design.

Exploiting Antigenic Diversity for Vaccine Design: THE CHLAMYDIA ArtJ PARADIGM*

We present an interdisciplinary approach that, by incorporating a range of experimental and computational techniques, allows the identification and characterization of functional/immunogenic domains. This approach has been applied to ArtJ, an arginine-binding protein whose orthologs in Chlamydiae trachomatis (CT ArtJ) and pneumoniae (CPn ArtJ) are shown to have different immunogenic properties despite a high sequence similarity (60% identity). We have solved the crystallographic structures of CT ArtJ and CPn ArtJ, which are found to display a type II transporter fold organized in two α-β domains with the arginine-binding region at their interface. Although ArtJ is considered to belong to the periplasm, we found that both domains contain regions exposed on the bacterial surface. Moreover, we show that recombinant ArtJ binds to epithelial cells in vitro, suggesting a role for ArtJ in host-cell adhesion during Chlamydia infection. Experimental epitope mapping and computational analysis of physicochemical determinants of antibody recognition revealed that immunogenic epitopes reside mainly in the terminal (D1) domain of both CPn and CT ArtJ, whereas the surface properties of the respective binding-prone regions appear sufficiently different to assume divergent immunogenic behavior. Neutralization assays revealed that sera raised against CPn ArtJ D1 partially reduce both CPn and CT infectivity in vitro, suggesting that functional antibodies directed against this domain may potentially impair chlamydial infectivity. These findings suggest that the approach presented here, combining functional and structure-based analyses of evolutionary-related antigens can be a valuable tool for the identification of cross-species immunogenic epitopes for vaccine development.

Structural Determinants of the Unusual Helix Stability of a De Novo Engineered Vascular Endothelial Growth Factor (VEGF) Mimicking Peptide

Understanding how an amino acid sequence folds into a well organized three-dimensional structure remains a challenge. The interest in protein folding comes from the possibility to predict the protein structure from genome-derived sequence, design proteins with new fold and understand protein misfolding. Peptide helix is a simple model system in which various contributions to helix formation can be dissected and understood qualitatively. Many strategies have been pursued to design peptide helices and notable results have been achieved even with very short sequences, but mainly these methods rely on the use of nonnatural amino acids or introducing constraints. In this paper, we report on the stability characterization, using CD, NMR and MD studies, of a designed, a-helical, 15-mer peptide (named QK), composed only of natural amino acids (sequence AcKLTWQELYQLKYKGI-NH2), which activates the VEGFdependent angiogenic response. The QK peptide shows an unusual thermal stability, whose structural determinants have been determined. These results could have implication in the field of protein folding and in the design of helical structured scaffolds for the realization of peptides for applications in chemical biology. As recently described, the NMR structure of QK in pure water presents a central helical sequence (residues 4–12), which corresponds to the VEGF N-terminal helix (residues 17–25), flanked by Nand C-capping regions. The helical conformation of QK represents an important prerequisite for its biological activity, since the isolated peptide, corresponding to the helix region of VEGF, does not assume a helical conformation and does not have significant biological activity. Interestingly, QK represents one of the very few examples of bioactive helical designed peptides, composed of only natural amino acids. To gain an insight into the molecular determinants of QK helical propensity, we examined the effect of the temperature on the QK structure through NMR and CD analyses. Primarily, the aggregation state of the peptide under conditions identical to those used in the NMR structure determination was confirmed by NMR DOSY experiments (see Supporting Information). The DOSY-derived diffusion coefficient value of 1.98@10 10 ms 1 is consistent with a QK monomer state. QK structure variations upon temperature increase were followed by TOCSY experiments. In the 298– 343 K range only small changes of the backbone chemical shifts were observed (Table 1 Supporting Information). The temperature dependences of Ha chemical shift deviations from the random coil values (DdHa) are reported in Figure 1a. Unusually, the chemical shift index (CSI) analysis indicates that at 343 K the peptide retains at least the 80% of the helix conformation at 298 K and the slight reduction occurs uniformly in 4–12 region (Figure 1a). The thermal behavior was also analyzed by CD spectroscopy which allowed [a] D. Diana, Prof. Dr. R. Fattorusso Dipartimento di Scienze Ambientali, Seconda UniversitC di Napoli via Vivaldi 43, 81100 Caserta (Italy) Fax: (+39)0823-274605 E-mail : roberto.fattorusso@unina2.it [b] B. Ziaco, Prof. Dr. C. Pedone, Dr. L. D. DIAndrea Istituto di Biostrutture e Bioimmagini, CNR, via Mezzocannone, 16 80134 Napoli (Italy) Fax: (+39)081-2534574 E-mail : ldandrea@unina.it [c] Dr. G. Colombo, Dr. G. Scarabelli Istituto di Chimica del Riconoscimento Molecolare, CNR via Bianco, 9, 20131 Milano (Italy) [d] Dr. A. Romanelli Dipartimento delle Scienze Biologiche UniversitC di Napoli “Federico II” via Mezzocannone 16, 80134 Napoli (Italy) Supporting information for this article is available on the WWW under http://www.chemistry.org or from the author: Peptide synthesis, circular dichroism, nmr spectroscopy and molecular dynamic simulations.

Mapping the structural and dynamical features of kinesin motor domains.

Kinesin motor proteins drive intracellular transport by coupling ATP hydrolysis to conformational changes that mediate directed movement along microtubules. Characterizing these distinct conformations and their interconversion mechanism is essential to determining an atomic-level model of kinesin action. Here we report a comprehensive principal component analysis of 114 experimental structures along with the results of conventional and accelerated molecular dynamics simulations that together map the structural dynamics of the kinesin motor domain. All experimental structures were found to reside in one of three distinct conformational clusters (ATP-like, ADP-like and Eg5 inhibitor-bound). These groups differ in the orientation of key functional elements, most notably the microtubule binding α4–α5, loop8 subdomain and α2b-β4-β6-β7 motor domain tip. Group membership was found not to correlate with the nature of the bound nucleotide in a given structure. However, groupings were coincident with distinct neck-linker orientations. Accelerated molecular dynamics simulations of ATP, ADP and nucleotide free Eg5 indicate that all three nucleotide states could sample the major crystallographically observed conformations. Differences in the dynamic coupling of distal sites were also evident. In multiple ATP bound simulations, the neck-linker, loop8 and the α4–α5 subdomain display correlated motions that are absent in ADP bound simulations. Further dissection of these couplings provides evidence for a network of dynamic communication between the active site, microtubule-binding interface and neck-linker via loop7 and loop13. Additional simulations indicate that the mutations G325A and G326A in loop13 reduce the flexibility of these regions and disrupt their couplings. Our combined results indicate that the reported ATP and ADP-like conformations of kinesin are intrinsically accessible regardless of nucleotide state and support a model where neck-linker docking leads to a tighter coupling of the microtubule and nucleotide binding regions. Furthermore, simulations highlight sites critical for large-scale conformational changes and the allosteric coupling between distal functional sites.

Kinesin-5 Allosteric Inhibitors Uncouple the Dynamics of Nucleotide, Microtubule, and Neck-Linker Binding Sites

Kinesin motor domains couple cycles of ATP hydrolysis to cycles of microtubule binding and conformational changes that result in directional force and movement on microtubules. The general principles of this mechanochemical coupling have been established; however, fundamental atomistic details of the underlying allosteric mechanisms remain unknown. This lack of knowledge hampers the development of new inhibitors and limits our understanding of how disease-associated mutations in distal sites can interfere with the fidelity of motor domain function. Here, we combine unbiased molecular-dynamics simulations, bioinformatics analysis, and mutational studies to elucidate the structural dynamic effects of nucleotide turnover and allosteric inhibition of the kinesin-5 motor. Multiple replica simulations of ATP-, ADP-, and inhibitor-bound states together with network analysis of correlated motions were used to create a dynamic protein structure network depicting the internal dynamic coordination of functional regions in each state. This analysis revealed the intervening residues involved in the dynamic coupling of nucleotide, microtubule, neck-linker, and inhibitor binding sites. The regions identified include the nucleotide binding switch regions, loop 5, loop 7, α4-α5-loop 13, α1, and β4-β6-β7. Also evident were nucleotide- and inhibitor-dependent shifts in the dynamic coupling paths linking functional sites. In particular, inhibitor binding to the loop 5 region affected β-sheet residues and α1, leading to a dynamic decoupling of nucleotide, microtubule, and neck-linker binding sites. Additional analyses of point mutations, including P131 (loop 5), Q78/I79 (α1), E166 (loop 7), and K272/I273 (β7) G325/G326 (loop 13), support their predicted role in mediating the dynamic coupling of distal functional surfaces. Collectively, our results and approach, which we make freely available to the community, provide a framework for explaining how binding events and point mutations can alter dynamic couplings that are critical for kinesin motor domain function.

Structural Analysis of a Helical Peptide Unfolding Pathway

The analysis of the folding mechanism in peptides adopting well-defined secondary structure is fundamental to understand protein folding. Herein, we describe the thermal unfolding of a 15-mer vascular endothelial growth factor mimicking alpha-helical peptide (QK(L10A)) through the combination of spectroscopic and computational analyses. In particular, on the basis of the temperature dependencies of QK(L10A) H(alpha) chemical shifts we show that the first phase of the thermal helix unfolding, ending at around 320 K, involves mainly the terminal regions. A second phase of the transition, ending at around 333 K, comprises the central helical region of the peptide. The determination of high-resolution QK(L10A) conformational preferences in water at 313 K allowed us to identify, at atomic resolution, one intermediate of the folding-unfolding pathway. Molecular dynamics simulations corroborate experimental observations detecting a stable central helical turn, which represents the most probable site for the helix nucleation in the folding direction. The data presented herein allows us to draw a folding-unfolding picture for the small peptide QK(L10A) compatible with the nucleation-propagation model. This study, besides contributing to the basic field of peptide helix folding, is useful to gain an insight into the design of stable helical peptides, which could find applications as molecular scaffolds to target protein-protein interactions.

A structural model for microtubule minus-end recognition and protection by CAMSAP proteins

CAMSAP and Patronin family members regulate microtubule minus-end stability and localization and thus organize noncentrosomal microtubule networks, which are essential for cell division, polarization and differentiation. Here, we found that the CAMSAP C-terminal CKK domain is widely present among eukaryotes and autonomously recognizes microtubule minus ends. Through a combination of structural approaches, we uncovered how mammalian CKK binds between two tubulin dimers at the interprotofilament interface on the outer microtubule surface. In vitro reconstitution assays combined with high-resolution fluorescence microscopy and cryo-electron tomography suggested that CKK preferentially associates with the transition zone between curved protofilaments and the regular microtubule lattice. We propose that minus-end-specific features of the interprotofilament interface at this site serve as the basis for CKKs minus-end preference. The steric clash between microtubule-bound CKK and kinesin motors explains how CKK protects microtubule minus ends against kinesin-13-induced depolymerization and thus controls the stability of free microtubule minus ends.

Mapping the Processivity Determinants of the Kinesin-3 Motor Domain

Kinesin superfamily members play important roles in many diverse cellular processes, including cell motility, cell division, intracellular transport, and regulation of the microtubule cytoskeleton. How the properties of the family-defining motor domain of distinct kinesins are tailored to their different cellular roles remains largely unknown. Here, we employed molecular-dynamics simulations coupled with energetic calculations to infer the family-specific interactions of kinesin-1 and kinesin-3 motor domains with microtubules in different nucleotide states. We then used experimental mutagenesis and single-molecule motility assays to further assess the predicted residue-wise determinants of distinct kinesin-microtubule binding properties. Collectively, our results identify residues in the L8, L11, and α6 regions that contribute to family-specific microtubule interactions and whose mutation affects motor-microtubule complex stability and processive motility (the ability of an individual motor to take multiple steps along its microtubule filament). In particular, substitutions of prominent kinesin-3 residues with those found in kinesin-1, namely, R167S/H171D, K266D, and R346M, were found to decrease kinesin-3 processivity 10-fold and thus approach kinesin-1 levels.

A posttranslational modification of the mitotic kinesin Eg5 that enhances its mechanochemical coupling and alters its mitotic function

Significance Members of the kinesin superfamily serve a wide variety of functions, and a dominant narrative for these molecular motors has been that each member of the superfamily is uniquely specialized to serve a very limited set of functions. However, it is now appreciated that many members of this group serve several distinct physiological roles, and it has been unclear how these kinesins accomplish this functional flexibility. In this report, we describe a posttranslational modification of the kinesin 5 family member Eg5 that dramatically alters its chemomechanical behavior to make it function much more efficiently under load and in ensembles. This work provides the biophysical context required to mechanistically understand the effects of modified Eg5 in dividing cells. Numerous posttranslational modifications have been described in kinesins, but their consequences on motor mechanics are largely unknown. We investigated one of these—acetylation of lysine 146 in Eg5—by creating an acetylation mimetic lysine to glutamine substitution (K146Q). Lysine 146 is located in the α2 helix of the motor domain, where it makes an ionic bond with aspartate 91 on the neighboring α1 helix. Molecular dynamics simulations predict that disrupting this bond enhances catalytic site–neck linker coupling. We tested this using structural kinetics and single-molecule mechanics and found that the K146Q mutation increases motor performance under load and coupling of the neck linker to catalytic site. These changes convert Eg5 from a motor that dissociates from the microtubule at low load into one that is more tightly coupled and dissociation resistant—features shared by kinesin 1. These features combined with the increased propensity to stall predict that the K146Q Eg5 acetylation mimetic should act in the cell as a “brake” that slows spindle pole separation, and we have confirmed this by expressing this modified motor in mitotically active cells. Thus, our results illustrate how a posttranslational modification of a kinesin can be used to fine tune motor behavior to meet specific physiological needs.

Mapping the Structural and Dynamical Features of Kinesin Proteins

Kinesin motor proteins drive intracellular transport by coupling ATP hydrolysis to conformational changes and directed movement along microtubules. Characterizing distinct conformations and their interconversion mechanism is thus essential to determining an atomic-level model of kinesin action. Here we report a comprehensive principal component analysis of 114 experimental structures along with the results of accelerated molecular dynamics simulations that together map the structural and dynamical features of the kinesin motor domain. All experimental structures were found to reside in one of eight distinct conformational clusters comprising two major groups. These groups differ in the orientation of key functional elements, including the microtubule binding alpha4-alpha5 subdomain. Group membership was found not to correlate with the nature of the bound nucleotide in a given structure. Accelerated molecular dynamics simulations of ATP, ADP and nucleotide free Eg5 indicated that all three nucleotide states could sample the major crystallographically observed conformations. Differences in the dynamic coupling of distal sites were evident in the simulations. In the ATP and APO simulations the neck-linker, loop8 and the alpha4-alpha5 subdomain display correlated motions that are absent in ADP simulations. Additional simulations predict that mutations G325A and G326A reduce the flexibility of these regions and disrupt their couplings. Furthermore, only in ADP simulations was the neck-linker region observed to undock. Additional APO simulations, commenced with an undocked neck-linker, formed coordinations reminiscent of the docked state. These interactions were absent in simulations of I359A mutants. Our combined results indicate that the reported ATP and ADP-like conformations of kinesin are intrinsically accessible regardless of nucleotide state. Furthermore, simulations highlight sites critical for large-scale conformational changes. We expect that further application of these methods will provide a framework for understanding the complete sequence of conformational changes and their relation to kinesins ATPase cycle.