Shi-Zhong Luo

Role of peptide self-assembly in antimicrobial peptides†

Antimicrobial peptides (AMPs) are considered as potential antibiotic substitutes because of their potent activities. Previous studies mainly focused on the effects of peptide charges and secondary structures, but the self‐assembly of AMPs was neglected. As more and more researchers notice the roles of peptide self‐assembly in AMPs, it has been considered as another important property. In this review, we will discuss the influences of peptide self‐assembly on the activity and mode of action, and some specific features it introduces to the AMPs, such as particular responsiveness, improved cell selectivity and stability and sustained release. In addition, some methods to design self‐assembling AMPs are primarily discussed. With further understanding about the self‐assembling regularity, design of particular self‐assembling AMPs will be very helpful for their applications, especially in the fields of drug delivery and biomedical engineering. Copyright

Insights into the Packing Switching of the EphA2 Transmembrane Domain by Molecular Dynamic Simulations

Receptor tyrosine kinases play an important role in mediating cell migration and adhesion associated with various biology processes. With a single-span transmembrane domain (TMD), the activities of the receptors are regulated by the definite packing configurations of the TMDs. For the EphA2 receptor, increasing studies have been conducted to investigate the packing domains that induce its switching TMD dimerization. However, the inherent transformation mechanisms including the interrelations among the involved packing domains remain unclear. Herein, we applied multiple simulation methods to explore the underlying packing mechanisms within the EphA2 TMD dimer. Our results demonstrated that the G(540)xxxG(544) contributed to the formation of the right-handed configuration while the heptad repeat L(535)xxxG(539)xxA(542)xxxV(546)xxL(549)xxxG(553) motif together with the FFxH(559) region mediated the parallel mode. Furthermore, the FF(557) residues packing mutually as rigid riveting structures were found comparable to the heptad repeat motif in maintaining the parallel configuration. In addition, the H(559) residue associated definitely with the lower bilayer leaflet, which was proved to stabilize the parallel mode significantly. The simulations provide a full range of insights into the essential packing motifs or residues involved in the switching TMD dimer configurations, which can enrich our comprehension toward the EphA2 receptor.

Dimerization and Structural Stability of Amyloid Precursor Proteins Affected by the Membrane Microenvironments

The lipid raft microenvironment is implicated in the generation of the pathological amyloid-β (Aβ) species in amyloid precursor protein (APP) that is associated with neurodegenerative diseases. Evidence shows that APP forms a transmembrane homodimer with changeable structures as a function of the membrane compositions. However, the molecular responsibility of the dimerization and structural alteration for the amyloidogenic process in segregated membranes remains largely unclear. Here, we performed multiple coarse grained (CG) simulations to explore the behavioral preference of the transmembrane domain of APP (called C99) that is affected by the lipid raft microenvironment. The results showed that C99 was anchored at the boundary of the lipid raft relying on the conserved hydrophobic motif of V710xxA713xxxV717xxxV721. Moreover, the dimerization of C99 was greatly destabilized by the lipid raft, which led to a susceptible switching packing conformation. The molecular driving forces were derived from the combined regulation of the saturated lipids and cholesterols rather than from the simple binding competition of cholesterol in the C99 dimerization. The molecular details of the differential dimerization in the raft-forming and bulk fluid bilayer environments were compared, and the structural information was helpful for further understanding the enzymolysis responsiveness of APP.

Self-assembly nanostructure controlled sustained release, activity and stability of peptide drugs

Peptides are considered as a new generation of drugs due to their high structural and functional diversity. However, the development of peptide drugs is always limited by their poor stability and short circulation time. Carriers are applied for peptide drug delivery, but there may be problems like poor loading efficiency and undesired xenobiotic toxicity. Peptide self-assembly is an effective approach to improve the stability and control the release of peptide drugs. In this study, two self-assembling anticancer peptides are designed by appending a pair of glutamic acid and asparagine to either the N-terminus or the C-terminus of a lytic peptide. This simple, yet rational sequence modification was made to change the amphiphilic pattern and secondary structural content of the parent peptide, thereby modulating its self-assembly process. It was found that the N-terminus modified peptide favors the formation of nanofibrils and the peptide with C-terminal modification formed micelles. Although both nanostructures showed prolonged action profiles and improved serum stability compared to the parent peptide, the morphology of the nanostructures is highly critical to manipulate the release profile of the free peptide from the assembly and regulate their bioactivity. We believe the self-assembly approach demonstrated in this study can be applied to a variety of therapeutic peptide drugs to improve their stability and therapeutic activity for the development of carrier-free drug delivery system.

From a pro-apoptotic peptide to a lytic peptide: One single residue mutation

Further discovery and design of new anticancer peptides are important for the development of anticancer therapeutics, and study on the detailed acting mechanism and structure-function relationship of peptides is critical for anticancer peptide design and application. In this study, a novel anticancer peptide ZXR-1 (FKIGGFIKKLWRSKLA) derived from a known anticancer peptide mauriporin was developed, and a mutant ZXR-2 (FKIGGFIKKLWRSLLA) with only one residue difference at the 14th position (Lys→Leu) was also engineered. Replacement of the lysine with leucine made ZXR-2 more potent than ZXR-1 in general. Even with only one residue mutation, the two peptides displayed distinct anticancer modes of action. ZXR-1 could translocate into cells, target on the mitochondria and induce cell apoptosis, while ZXR-2 directly targeted on the cell membranes and caused membrane lysis. The variance in their acting mechanisms might be due to the different amphipathicity and positive charge distribution. In addition, the two Ile-Leu pairs (3-10 and 7-14) in ZXR-2 might also play a role in improving its cytotoxicity. Further study on the structure-function relationship of the two peptides may be beneficial for the design of novel anticancer peptides and peptide based therapeutics.

Molecular Dynamic Simulation of the Self-Assembly of DAP12-NKG2C Activating Immunoreceptor Complex

The DAP12-NKG2C activating immunoreceptor complex is one of the multisubunit transmembrane protein complexes in which ligand-binding receptor chains assemble with dimeric signal-transducing modules through non-covalent associations in their transmembrane (TM) domains. In this work, both coarse grained and atomistic molecular dynamic simulation methods were applied to investigate the self-assembly dynamics of the transmembrane domains of the DAP12-NKG2C activating immunoreceptor complex. Through simulating the dynamics of DAP12-NKG2C TM heterotrimer and point mutations, we demonstrated that a five-polar-residue motif including: 2 Asps and 2 Thrs in DAP12 dimer, as well as 1 Lys in NKG2C TM plays an important role in the assembly structure of the DAP12-NKG2C TM heterotrimer. Furthermore, we provided clear evidences to exclude the possibility that another NKG2C could stably associate with the DAP12-NKG2C heterotrimer. Based on the simulation results, we proposed a revised model for the self-assembly of DAP12-NKG2C activating immunoreceptor complex, along with a plausible explanation for the association of only one NKG2C with a DAP12 dimer.

Critical Residues and Motifs for Homodimerization of the First Transmembrane Domain of the Plasma Membrane Glycoprotein CD36

The plasma transmembrane (TM) glycoprotein CD36 is critically involved in many essential signaling processes, especially the binding/uptake of long-chain fatty acids and oxidized low-density lipoproteins. The association of CD36 potentially activates cytosolic protein tyrosine kinases that are thought to associate with the C-terminal cytoplasmic tail of CD36. To understand the mechanisms by which CD36 mediates ligand binding and signal transduction, we have characterized the homo-oligomeric interaction of CD36 TM domains in membrane environments and with molecular dynamics (MD) simulations. Analysis of pyrene- and coumarin-labeled TM1 peptides in SDS by FRET confirmed the homodimerization of the CD36 TM1 peptide. Homodimerization assays of CD36 TM domains with the TOXCAT technique showed that its first TM (TM1) domain, but not the second TM (TM2) domain, could homodimerize in a cell membrane. Small-residue, site-specific mutation scanning revealed that the CD36 TM1 dimerization is mediated by the conserved small residues Gly12, Gly16, Ala20, and Gly23. Furthermore, molecular dynamics (MD) simulation studies demonstrated that CD36 TM1 exhibited a switching dimerization with two right-handed packing modes driven by the 12GXXXGXXXA20 and 20AXXG23 motifs, and the mutational effect of G16I and G23I revealed these representative conformations of CD36 TM1. This packing switch pattern of CD36 TM1 homodimer was further examined and confirmed by FRET analysis of monobromobimane (mBBr)-labeled CD36 TM1 peptides. Overall, this work provides a structural basis for understanding the role of TM association in regulating signal transduction via CD36.

How charge distribution influences the function of membrane-active peptides: Lytic or cell-penetrating?

Lytic and cell-penetrating peptides (CPPs) are both membrane-active peptides sharing similar physicochemical properties. Although their respective functions have been intensively investigated, the difference of intrinsic properties between these two types of peptides is rarely discussed. In this study, we designed a series of analogs of a recently discovered CPP ZXR-1 (FKIGGFIKKLWRSKLA) by varying the charge distributions both on the helical wheel projection and along the sequence. These peptides showed different functions on cell membranes, including membrane lytic (peptide Z1), cell-penetrating (peptide ZXR-1, Z2 and Z3), and inactive (peptide Z4) peptides. The three groups of peptides displayed different interactions with model lipid monolayer, and found that peptide insertion might be an important dynamic step to distinguish lytic and cell penetrating functions. Based on the analysis of charge distribution patterns, it was proposed that the charge distributions on the helical wheel and along the sequence are both able to influence the functions of the membrane-active peptides. This finding provides a further understanding about the effect of charge distribution on the functions of membrane-active peptides, and will be helpful for the design of functional peptides.

Insights into the transmembrane helix associations of kit ligand by molecular dynamics simulation and TOXCAT

Kit ligand (KITL) plays important roles in cell proliferation, differentiation, and survival via interaction with its receptor Kit. The previous studies demonstrated that KITL formed a noncovalent homodimer through transmembrane (TM) domain; however, the undergoing mechanism of transmembrane association that determines KITL TM dimerization is still not clear. Herein, molecular dynamics (MD) simulation strategy and TOXCAT assay were combined to characterize the dimerization interface and structure of KITL TM in details. KITL TM formed a more energetically favorable noncovalent dimer through a conserved SxxxGxxxG motif in the MD simulation. Furthermore, the TOXCAT results demonstrated that KITL TM self‐associated strongly in the bilayer membrane environment. Mutating any one of the small residues Ser11, Gly15 or Gly19 to Ile disrupted KITL TM dimerization dramatically, which further validated our MD simulation results. In addition, our results showed that Tyr22 could help to stabilize the TM interactions via interacting with the phosphoric group in the bilayer membrane. Pro7 did not induce helix kinks or swivel angles in KITL TM, but it was related with the pitch of the turn around this residue so as to affect the dimer formation. Combining the results of computer modeling and experimental mutagenesis studies on the KITL TM provide new insights for the transmembrane helix association of KITL dimerization. Proteins 2017; 85:1362–1370.

High-Resolution Insights into the Stepwise Self-Assembly of Nanofiber from Bioactive Peptides

Peptide self-assembly has a profound biological significance since self-assembled bioactive peptides are gifted with improved bioactivity as well as life-span. In this study, peptide self-assembly was investigated using a therapeutic peptide, PTP-7S (EENFLGALFKALSKLL). Combining experiments of atomic force microscopy (AFM), circular dichroism (CD), and 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence spectra, PTP-7S showed the α-helical structure and was found self-assembling into nanofibers in solution. Relying on the coarse-grained (CG) dynamic simulations, the self-assembling of PTP-7S was revealed as a stepwise process that peptide monomers first clustered into peptide-assembling units (PUs) with charged surface, and then the PUs integrated together to construct nanofibril aggregates. Different roles of the nonbonded driving forces did play in the two phases: the hydrophobic force and electrostatic interaction acted as the predominant motivations in the formation of PUs and nanofiber, respectively. Moreover, the electrostatic interaction helped to guide the longitudinal growth of peptide nanofibers. A sequence principle is proposed for peptide self-assembling in aqueous solution: a balance of the counter charges and sufficient hydrophobicity degree. The self-assembled PTP-7S displayed good anticancer activity, proteases resistance, and sustained drug-release, showing a great potential for clinical application. This study reveals the molecular mechanism in explaining PTP-7S self-assembly and it is beneficial for future innovation of the self-assembled bioactive peptides.