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PG16 is a broadly neutralizing antibody that binds to the gp120 subunit of the HIV-1 Env protein. The major interaction site is formed by the unusually long complementarity determining region (CDR) H3. The CDRH3 residue Tyr100H is known to represent a tyrosine sulfation site; however, this modification is not present in the experimental complex structure of PG16 with full-length HIV-1 Env. To investigate the role of sulfation for this complex, we modeled the sulfation of Tyr100H and compared the dynamics and energetics of the modified and unmodified complex by molecular dynamics simulations at the atomic level. Our results show that sulfation does not affect the overall conformation of CDRH3, but still enhances gp120 interactions both at the site of modification and for the neighboring residues. This stabilization affects not only protein–protein contacts, but also the interactions between PG16 and the gp120 glycan shield. Furthermore, we also investigated whether PG16-CDRH3 is a suitable template for the development of peptide mimetics. For a peptide spanning residues 93-105 of PG16, we obtained an experimental EC50 value of 3nm for the binding of gp120 to the peptide. This affinity can be enhanced by almost one order of magnitude by artificial disulfide bonding between residues 99 and 100F. In contrast, any truncation results in significantly lower affinity, suggesting that the entire peptide segment is involved in gp120 recognition. Given their high affinity, it should be possible to further optimize the PG16-derived peptides as potential inhibitors of HIV invasion.

The family of neuropeptide Y (NPY) receptors comprises four subtypes (Y1R, Y2R, Y4R, Y5R), which are addressed by at least three endogenous peptides, i.e., NPY, peptide YY, and pancreatic polypeptide (PP), the latter showing a preference for Y4R. A series of cyclic oligopeptidic Y4R ligands were prepared by applying a novel approach, i.e., N-terminus to arginine side-chain cyclization. Most peptides acted as Y4R partial agonists, showing up to 60-fold higher Y4R affinity compared to the linear precursor peptides. Two cyclic hexapeptides (18, 24) showed higher Y4R potency (Ca2+ aequorin assay) and, with pKi values >10, also higher Y4R affinity compared to human pancreatic polypeptide (hPP). Compounds such as 18 and 24, exhibiting considerably lower molecular weight and considerably more pronounced Y4R selectivity than PP and previously described dimeric peptidic ligands with high Y4R affinity, represent promising leads for the preparation of labeled tool compounds and might support the development of drug-like Y4R ligands.

The family of neuropeptide Y (NPY) receptors comprises four subtypes (Y

G protein-coupled receptors (GPCRs) are a main drug target and therefore a hot topic in pharmaceutical research. One important prerequisite to understand how a certain ligand affects a GPCR is precise knowledge about its binding mode and the specific underlying interactions. If no crystal structure of the respective complex is available, computational methods can be used to deduce the binding site. One of them are metadynamics simulations which have the advantage of an enhanced sampling compared to conventional molecular dynamics simulations. However, the enhanced sampling of higher-energy states hampers identification of the preferred binding mode. Here, we present a novel protocol based on clustering of multiple walker metadynamics simulations which allows identifying the preferential binding mode from such conformational ensembles. We tested this strategy for three different model systems namely the histamine H1 receptor in combination with its physiological ligand histamine, as well as the &beta, 2 adrenoceptor with its agonist adrenaline and its antagonist alprenolol. For all three systems, the proposed protocol was able to reproduce the correct binding mode known from the literature suggesting that the approach can more generally be applied to the prediction of GPCR ligand binding in future.

Alzheimer's disease is characterized by the aggregation of Amyloid-β (Aβ) peptide into oligomers, fibrils and plaques. Many factors influencing this process as well as the stability of the various Aβ aggregates are known to date, and include the concentration and type of metal ions. Most experimental and theoretical studies have concentrated on heavy metal ions, like Fe2+, Zn2+, or Cu2+, while the smaller alkali ions Li+, Na+, and K+ have not gained much attention notwithstanding their role and ubiquity in physiological environments. In this work, we applied atomistic molecular dynamics simulations to investigate the potential role of these alkali ions in stabilizing fibrillar Aβ oligomers of different size and topology, i.e., single and double filament systems comprising 3-24 peptide chains per filament. We find a pronounced difference on the molecular level in the interaction behavior with free carboxylate groups of the Aβ oligomer: Li+ forms stable bridged interactions, whereas K+ interacts more transiently and lacks bridging. The behavior of Na+ is in between, so that this ion-protein interaction obeys the renowned Hofmeister series. These differences are also reflected in the ability of the alkali ions to stabilize the oligomer secondary structure. The stabilizing effect is most pronounced for the smaller fibrillar oligomers, suggesting that the type of alkali ion critically affects the initial stages of fibril formation. Our findings thus offer a molecular explanation for the observation that the polymorphisms of Aβ fibril structures are caused by differences in the surrounding ionic environment. Graphical abstract Influence of alkali ions on the structure and stability of fibrillar amyloid-β oligomers.

Purpose\ud Microcephaly is a sign of many genetic conditions but has been rarely systematically evaluated. We therefore comprehensively studied the clinical and genetic landscape of an unselected cohort of patients with microcephaly.\ud Methods\ud We performed clinical assessment, high-resolution chromosomal microarray analysis, exome sequencing, and functional studies in 62 patients (58% with primary microcephaly [PM], 27% with secondary microcephaly [SM], and 15% of unknown onset).\ud Results\ud We found severity of developmental delay/intellectual disability correlating with severity of microcephaly in PM, but not SM. We detected causative variants in 48.4% of patients and found divergent inheritance and variant pattern for PM (mainly recessive and likely gene-disrupting [LGD]) versus SM (all dominant de novo and evenly LGD or missense). While centrosome-related pathways were solely identified in PM, transcriptional regulation was the most frequently affected pathway in both SM and PM. Unexpectedly, we found causative variants in different mitochondria-related genes accounting for ~5% of patients, which emphasizes their role even in syndromic PM. Additionally, we delineated novel candidate genes involved in centrosome-related pathway (SPAG5, TEDC1), Wnt signaling (VPS26A, ZNRF3), and RNA trafficking (DDX1).\ud Conclusion\ud Our findings enable improved evaluation and genetic counseling of PM and SM patients and further elucidate microcephaly pathways.

Binding of histamine to the G-protein coupled histamine H1 receptor plays an important role in the context of allergic reactions; however, no crystal structure of the resulting complex is available yet. To deduce the histamine binding site, we performed unbiased molecular dynamics (MD) simulations on a microsecond time scale, which allowed to monitor one binding event, in which particularly the residues of the extracellular loop 2 were involved in the initial recognition process. The final histamine binding pose in the orthosteric pocket is characterized by interactions with Asp1073.32, Tyr1083.33, Thr1945.43, Asn1985.46, Trp4286.48, Tyr4316.51, Phe4326.52, and Phe4356.55, which is in agreement with existing mutational data. The conformational stability of the obtained complex structure was subsequently confirmed in 2 μs equilibrium MD simulations, and a metadynamics simulation proved that the detected binding site represents an energy minimum. A complementary investigation of a D107A mutant, which has experimentally been shown to abolish ligand binding, revealed that this exchange results in a significantly weaker interaction and enhanced ligand dynamics. This finding underlines the importance of the electrostatic interaction between the histamine ammonium group and the side chain of Asp1073.32 for histamine binding.

Synthetic analogues of mycobacterial trehalose-dimycolate such as trehalose acyl esters have been proposed as novel adjuvants for vaccination. They induce an immune response by binding to the macrophage C-type lectin receptor Mincle. The binding site of trehalose is known, but there is yet only very limited structural information about the binding mode of the acyl esters. Here, we performed a systematic molecular dynamics study of trehalose mono-and diesters with different chain lengths. All acyl chains investigated exhibited a high flexibility and interacted almost exclusively with a hydrophobic groove on Mincle. Despite the limited length of this hydrophobic groove, the distal parts of the longer monoesters can still form additional interactions with this surface region due to their conformational flexibility. In diesters, a certain length of the second acyl chain is required to contact the hydrophobic groove. However, a stable concomitant accommodation of both acyl chains in the groove is hampered by the conformational rigidity of Mincle. Instead, multiple dynamic interaction modes are observed, in which the second acyl chain contributes to binding. This detailed structural information is considered helpful for the future design of more affine ligands that may foster the development of novel adjuvants.

Mature B cells express immunoglobulin M (IgM)- and IgD-isotype B cell antigen receptors, but the importance of IgD for B cell function has been unclear. By using a cellular in vitro system and corresponding mouse models, we found that antigens with low valence activated IgM receptors but failed to trigger IgD signaling, whereas polyvalent antigens activated both receptor types. Investigations of the molecular mechanism showed that deletion of the IgD-specific hinge region rendered IgD responsive to monovalent antigen, whereas transferring the hinge to IgM resulted in responsiveness only to polyvalent antigen. Our data suggest that the increased IgD/IgM ratio on conventional B-2 cells is important for preferential immune responses to antigens in immune complexes, and that the increased IgM expression on B-1 cells is essential for B-1 cell homeostasis and function.

Synthetic analogues of mycobacterial trehalose-dimycolate such as trehalose acyl esters have been proposed as novel adjuvants for vaccination. They induce an immune response by binding to the macrophage C-type lectin receptor Mincle. The binding site of trehalose is known, but there is yet only very limited structural information about the binding mode of the acyl esters. Here, we performed a systematic molecular dynamics study of trehalose mono-and diesters with different chain lengths. All acyl chains investigated exhibited a high flexibility and interacted almost exclusively with a hydrophobic groove on Mincle. Despite the limited length of this hydrophobic groove, the distal parts of the longer monoesters can still form additional interactions with this surface region due to their conformational flexibility. In diesters, a certain length of the second acyl chain is required to contact the hydrophobic groove. However, a stable concomitant accommodation of both acyl chains in the groove is hampered by the conformational rigidity of Mincle. Instead, multiple dynamic interaction modes are observed, in which the second acyl chain contributes to binding. This detailed structural information is considered helpful for the future design of more affine ligands that may foster the development of novel adjuvants.

In their contribution Verma et al. propose a drastic anti-cancer effect of quercetin and taxifolin based on molecular dynamics (MD) simulations of the MDM2-p53 protein complex bound to a ligand. However, we have severe concerns that the observed dissociation of the MDM2-p53 complex is not caused by ligand interaction, but an artifact arising from technical issues of post-processing the simulation data. This becomes evident from the root-mean-square deviation plots versus time of the two ligand-bound protein complex simulations, where huge and immediate jumps indicate analysis problems. In order to corroborate our view, we performed MD simulations of the MDM2-p53 complex without any ligand, which is experimentally known to be stable. We observed similar pseudo-dissociation events in the raw MD data that disappeared after proper post-processing. Thus, we strongly recommend a careful re-analysis of the original raw MD data by the authors. Although we do not exclude a dissociative effect of quercetin and taxifolin onto MDM2-p53, and therefore, potential anti-cancer properties of those compounds, the effect seen in the MD simulations should be much more subtle on that timescale.

The amyloid-β (Aβ) peptide is one key molecule in the pathogenesis of Alzheimer's disease. We investigated the conformational stability of a nonfibrillar tetrameric Aβ structure by molecular dynamics (MD) simulations revealing that the stability of the Aβ tetramer depends critically on the C-terminal length. In contrast to the Aβ17-40 tetramer, which proved to be instable, the simulations demonstrate structural integrity of the Aβ17-42 and Aβ17-43 tetramers. These differences in stability can be attributed to an extension of the middle strand of a three-stranded antiparallel β sheet through residues 41-43, only present in the longer Aβ species that aggregate faster and are more neurotoxic. Additional MD simulations demonstrate that this higher stability is also present in the monomers forming the tetramer. In conclusion, our findings suggest the existence of a nonfibrillar oligomer topology that is significantly more stable for the longer Aβ species, thus offering a structural explanation for their higher neurotoxicity.

A variety of neurodegenerative disorders, including Alzheimer disease (AD), are associated with neurofibrillary tangles composed of the tau protein, as well as toxic tau oligomers. Inhibitors of pathological tau aggregation, interrupting tau self-assembly, might be useful for the development of therapeutics. Employing mirror image phage display with a large peptide library (over 109 different peptides), we have identified tau fibril binding peptides consisting of d-enantiomeric amino acids. d-enantiomeric peptides are extremely protease stable and not or less immunogenic than l-peptides, and the suitability of d-peptides for in vivo applications have already been demonstrated. Phage display selections were performed using fibrils of the d-enantiomeric hexapeptide VQIVYK, representing residues 306 to 311 of the tau protein, as a target. VQIVYK has been demonstrated to be important for fibril formation of the full lengths protein and forms fibrils by itself. Here, we report on d-enantiomeric peptides, which bind to VQIVYK, tau isoforms like tau3RD (K19) as well as to full lengths tau fibrils, and modulate the aggregation of the respective tau form. The peptides are able to penetrate cells and might be interesting for therapeutic and diagnostic applications in AD research.

The nuclear lamina lines the inner nuclear membrane providing a structural framework for the nucleus. Cellular processes, such as nuclear envelope breakdown during mitosis or nuclear export of large ribonucleoprotein complexes, are functionally linked to the disassembly of the nuclear lamina. In general, lamina disassembly is mediated by phosphorylation, but the precise molecular mechanism is still not completely understood. Recently, we suggested a novel mechanism for lamina disassembly during the nuclear egress of herpesviral capsids which involves the cellular isomerase Pin1. In this study, we focused on mechanistic details of herpesviral nuclear replication to demonstrate the general importance of Pin1 for lamina disassembly. In particular, Ser22-specific lamin phosphorylation consistently generates a Pin1-binding motif in cells infected with human and animal alpha-, beta-, and gammaherpesviruses. Using nuclear magnetic resonance spectroscopy, we showed that binding of Pin1 to a synthetic lamin peptide induces its cis/trans isomerization in vitro. A detailed bioinformatic evaluation strongly suggests that this structural conversion induces large-scale secondary structural changes in the lamin N-terminus. Thus, we concluded that a Pin1-induced conformational change of lamins may represent the molecular trigger responsible for lamina disassembly. Consistent with this concept, pharmacological inhibition of Pin1 activity blocked lamina disassembly in herpesvirus-infected fibroblasts and consequently impaired virus replication. In addition, a phospho-mimetic Ser22Glu lamin mutant was still able to form a regular lamina structure and overexpression of a Ser22-phosphorylating kinase did not induce lamina disassembly in Pin1 knockout cells. Intriguingly, this was observed in absence of herpesvirus infection proposing a broader importance of Pin1 for lamina constitution. Thus, our results suggest a functional model of similar events leading to disassembly of the nuclear lamina in response to herpesviral or inherent cellular stimuli. In essence, Pin1 represents a regulatory effector of lamina disassembly that promotes the nuclear pore-independent egress of herpesviral capsids., Author Summary Viruses often adopt preexisting cellular pathways to promote their own replication. In this regard, the recently discovered alternative mechanism for the nuclear export of large messenger ribonucleoprotein (mRNP) complexes is particularly noteworthy. This process is mechanistically similar to the nuclear egress of herpesviruses, which appear to utilize cellular pathways and effectors to release assembled capsids from the host nucleus. While vesicle formation and scission events at nuclear membranes are now increasingly understood in greater detail, the precise mechanism of the preceding disassembly of the nuclear lamina still awaits a defined molecular characterization. Here, we used herpesviruses in their property to induce a nucleocytoplasmic viral capsid export for our investigation of nuclear lamina disassembly. We identified a mechanism that promotes lamina disassembly by a conformational change of lamins, mediated by the cellular isomerase Pin1 in a phosphorylation-dependent manner. Intriguingly, Pin1 appeared to control the rearrangement of phosphorylated lamins and their transient displacement from the nuclear lamina. Our study suggests that Pin1 functions as a major regulatory effector of lamina disassembly and thus determines the nuclear egress pathway of herpesviruses.

Several lines of evidence suggest that the amyloid-β-peptide (Aβ) plays a central role in the pathogenesis of Alzheimer's disease (AD). Not only Aβ fibrils but also small soluble Aβ oligomers in particular are suspected to be the major toxic species responsible for disease development and progression. The present study reports on in vitro and in vivo properties of the Aβ targeting d-enantiomeric amino acid peptide D3. We show that next to plaque load and inflammation reduction, oral application of the peptide improved the cognitive performance of AD transgenic mice. In addition, we provide in vitro data elucidating the potential mechanism underlying the observed in vivo activity of D3. These data suggest that D3 precipitates toxic Aβ species and converts them into nonamyloidogenic, nonfibrillar, and nontoxic aggregates without increasing the concentration of monomeric Aβ. Thus, D3 exerts an interesting and novel mechanism of action that abolishes toxic Aβ oligomers and thereby supports their decisive role in AD development and progression.

Recent experimental data demonstrate that small, soluble amyloid-beta42 oligomers play an important role in Alzheimer's disease because they exhibit neurotoxic properties and also act as seed for fibril growth. We performed all-atom molecular dynamics simulations in explicit solvent of 0.7 micros in total on five Abeta9-42 oligomers (monomer through pentamer) starting from the fibril conformation. The initial conformation proves to be stable in the trimer to pentamer, and the two parallel in-register beta-sheets as well as the connecting turn are preserved. The dimer undergoes larger conformational changes in its C-terminus, and the predominant conformation detected exhibits an additional antiparallel beta-sheet in one of the subunits. This conformational rearrangement allows efficient shielding of hydrophobic residues from the solvent, which is not possible for a dimer in the fibril conformation. In addition to the presence of the hydrogen bonds in the beta-sheets, the larger oligomers are stabilized by interchain D23-K28 salt bridges, whereas a D23-N27 interaction is found in the dimer. The degree of structural similarity to the fibril conformation detected for the oligomers in the simulation may also offer a structural explanation for the experimental finding that trimers and tetramers act as more potent seeds in fibril formation than dimers because only small conformational changes will be required for fibril growth. The fact that the dimer predominantly exists in conformations distinct from the larger oligomers and the fibril is also interesting for the design of anti-Alzheimer drugs, because it suggests that multiple drugs might be required to target the structurally different neurotoxic oligomers.

Converging lines of evidence suggest that soluble A beta-amyloid oligomers play a pivotal role in the pathogenesis of Alzheimer's disease, and present direct effectors of synaptic and cognitive dysfunction. Three pathological E22-A beta-amyloid point mutants (E22G, E22K, E22Q) and the deletion mutant E22 Delta exhibit an enhanced tendency to form prefibrillar aggregates. The present study assessed the effect of these four mutations using molecular dynamics simulations and subsequent structural and energetic analyses. Our data shows that E22 plays a unique role in wild type A beta, since it has a destabilising effect on the oligomer structure due to electrostatic repulsion between adjacent E22 side chains. Mutations in which E22 is replaced by an uncharged residue result in higher oligomer stability. This effect is also observed to a lesser extent for the E22K mutation and is consistent with its lower pathogenicity compared to other mutants. Interestingly, deletion of E22 does not destroy the amyloid fold but is compensated by local changes in the backbone geometry that allow the preservation of a structurally important salt bridge. The finding that all mutant oligomers investigated exhibit higher internal stability than the wild type offers an explanation for the experimentally observed enhanced oligomer formation and stability.

Despite amyloid plaques, consisting of insoluble, aggregated amyloid-β peptides, being a defining feature of Alzheimer’s disease, their significance has been challenged due to controversial findings regarding the correlation of cognitive impairment in Alzheimer’s disease with plaque load. The amyloid cascade hypothesis defines soluble amyloid-β oligomers, consisting of multiple amyloid-β monomers, as precursors of insoluble amyloid-β plaques. Dissecting the biological effects of single amyloid-β oligomers, for example of amyloid-β dimers, an abundant amyloid-β oligomer associated with clinical progression of Alzheimer’s disease, has been difficult due to the inability to control the kinetics of amyloid-β multimerization. For investigating the biological effects of amyloid-β dimers, we stabilized amyloid-β dimers by an intermolecular disulphide bridge via a cysteine mutation in the amyloid-β peptide (Aβ-S8C) of the amyloid precursor protein. This construct was expressed as a recombinant protein in cells and in a novel transgenic mouse, termed tgDimer mouse. This mouse formed constant levels of highly synaptotoxic soluble amyloid-β dimers, but not monomers, amyloid-β plaques or insoluble amyloid-β during its lifespan. Accordingly, neither signs of neuroinflammation, tau hyperphosphorylation or cell death were observed. Nevertheless, these tgDimer mice did exhibit deficits in hippocampal long-term potentiation and age-related impairments in learning and memory, similar to what was observed in classical Alzheimer’s disease mouse models. Although the amyloid-β dimers were unable to initiate the formation of insoluble amyloid-β aggregates in tgDimer mice, after crossbreeding tgDimer mice with the CRND8 mouse, an amyloid-β plaque generating mouse model, Aβ-S8C dimers were sequestered into amyloid-β plaques, suggesting that amyloid-β plaques incorporate neurotoxic amyloid-β dimers that by themselves are unable to self-assemble. Our results suggest that within the fine interplay between different amyloid-β species, amyloid-β dimer neurotoxic signalling, in the absence of amyloid-β plaque pathology, may be involved in causing early deficits in synaptic plasticity, learning and memory that accompany Alzheimer’s disease.10.1093/brain/awv355_video_abstract awv355_video_abstract

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The recently introduced multipole approach for computing the molecular electrostatic potential (MEP) within the semiempirical neglect of diatomic differential overlap (NDDO) framework [Horn AHC, Lin Jr-H., Clark T (2005) Theor Chem Acc 114:159–168] has been used to obtain atomic charges of nearly ab initio quality by scaling the semiempirical MEP. The parameterization set comprised a total of 797 compounds and included not only the newly parameterized AM1* elements Al, Si, P, S, Cl, Ti, Zr, and Mo but also the standard AM1 elements H, C, N, O and F. For comparison, the ZDO-approximated MEP was also calculated analytically in the spd-basis. For the AM1*-optimized structures, single-point calculations at the B3LYP, HF and MP2 levels with the 6-31G(d) and LanL2DZP basis sets were performed to obtain the MEP. The regression analysis of all 12 combinations of semiempirical and ab initio MEP data yielded correlation coefficients of at least 0.99 in all cases. Scaling the analytical and multipole-derived semiempirical MEP by the regression coefficients yielded mean unsigned errors below 2.6 and 1.9 kcal mol−1, respectively. Subsequently, for 22 drug molecules from the World Drug Index, atomic charges were computed according to the RESP procedure using XX/6-31G(d) (XX=B3LYP, HF, MP2) and scaled AM1* multipole MEP; the correlation coefficients obtained are 0.83, 0.85 and 0.83, respectively. Figure: Schematic representation of the atomic charge generation: The molecular electrostatic potential (MEP) is calculated using the AM1* Hamiltonian; then the semiempirical MEP is scaled to DFT or ab initio level, and atomic charges are generated subsequently by the restraint electrostatic potential (RESP) fit method.

The prion protein is thought to induce prion diseases by changing its conformation from the cellular form, PrP(C), into the infectious Scrapie-form, PrP(Sc). Little is known about the structural and dynamical features of this conformational change. We here introduce a novel concept that involves rare large scale motions between the subdomains beta1-alpha1-beta2 and alpha2-alpha3 in the carboxy-terminal, globular part of PrP. The interface between these two subdomains carries most pathogenic mutations known to be associated with prion diseases. Based on computational simulations as well as experimental results we propose that such a large scale motion subsequently destabilizes large parts of the cellular conformer PrP(C), thus, rendering it prone to structural rearrangements, including aggregation of now partially unfolded parts of the PrP sequence. We hypothesize that such large scale motions occur as a rare event even under equilibrium conditions and that the interaction of such partially destabilized PrP(C)-conformers, which we named PrP(C*), contributes to the formation of pathogenic oligomeric species of the prion protein.

We report a consistent set of AMBER force-field parameters for the most common phosphorylated amino acids, phosphoserine, phosphothreonine, phosphotyrosine, and phosphohistidine in different protonation states. The calculation of atomic charges followed the original restrained electrostatic potential fitting procedure used to determine the charges for the parm94/99 parameter set, taking alpha-helical and beta-strand conformations of the corresponding ACE-/NME-capped model peptide backbone into account. Missing force-field parameters were taken directly from the general AMBER force field (gaff) and the parm99 data set with minor modifications, or were newly generated based on ab initio calculations for model systems. Final parameters were validated by geometry optimizations and molecular-dynamics simulations. Template libraries for the phosphorylated amino acids in Leap format and corresponding frcmod parameter files are made available. [Figure: see text].

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A major problem in the antiretroviral treatment of HIV-infections with protease-inhibitors is the emergence of resistance, resulting from the occurrence of distinct mutations within the protease molecule. In the present work molecular dynamics simulations of an active-site mutation (D30N) and a nonactive-site mutation (N88S) of HIV-1 protease that both directly confer resistance to the protease inhibitor Nelfinavir but not to Amprenavir were performed and compared to wild-type HIV-protease. A decreased interaction energy between protease and Nelfinavir was observed for the D30N mutant giving a plausible explanation for resistance, while the N88S mutation did not significantly affect the interaction energies in the bound form. Structural analysis including both ligand-bound and unliganded HIV-1 proteases revealed that the free N88S mutant protease shows significant differences in its hydrogen bonding pattern compared to free or Nelfinavir-bound wild-type protease. In particular, Asp30 forms more frequently a hydrogen bond with Ser88 in the unbound N88S mutant thus interfering with the Asp30-Nelfinavir interaction. These findings suggest that different molecular mechanisms contribute to resistance in active-site and nonactive-site mutants and propose a mechanism for the N88S mutant that is based on a shift of the conformational equilibrium of the unbound protease.

An extension of the AM1 semiempirical molecular orbital technique, AM1*, is introduced. AM1* uses AM1 parameters and theory unchanged for the elements H, C, N, O and F. The elements P, S and Cl have been reparameterized using an additional set of d orbitals in the basis set and with two-center core-core parameters, rather than the Gaussian functions used to modify the core-core potential in AM1. Voityuk and Rösch's AM1(d) parameters have been adopted unchanged for AM1* with the exception that new core-core parameters are defined for Mo-P, Mo-S and Mo-Cl interactions. Thus, AM1* gives identical results to AM1 for compounds with only H, C, N, O, and F, AM1(d) for compounds containing Mo, H, C, N, O and F only, but differs for molybdenum compounds containing P, S or Cl. The performance and typical errors of AM1* are discussed.

We discuss problems and features of current semiempirical molecular orbital techniques and test some of the approximations and assumptions used. Prerequisites for a ‘‘next generation’’ technique include orthogonalization corrections, effective core potentials and an implicit dispersion term. However, validation of experimental parameterization data using density functional theory or the Gaussian 2 approach reveals significant errors in some cases. Developers of future methods will need to validate all their parameterization data and may no longer be able to parameterize for heats of formation at 298 K, but may need to use Born–Oppenheimer binding energies. We also suggest that there is no inherent reason that semiempirical techniques should not reproduce hydrogen bonding and show that the Gaussian potentials added to the core–core terms in AM1 and the PMn methods actually weaken hydrogen bonding, rather than strengthening it.