Michael Lammers

Structural and mechanistic insights into the interaction between Rho and mammalian Dia.

Formins are involved in a variety of cellular processes that require the remodelling of the cytoskeleton. They contain formin homology domains FH1 and FH2, which initiate actin assembly. The Diaphanous-related formins form a subgroup that is characterized by an amino-terminal Rho GTPase-binding domain (GBD) and an FH3 domain, which bind somehow to the carboxy-terminal Diaphanous autoregulatory domain (DAD) to keep the protein in an inactive conformation. Upon binding of activated Rho proteins, the DAD is released and the ability of the formin to nucleate and elongate unbranched actin filaments is induced. Here we present the crystal structure of RhoC in complex with the regulatory N terminus of mammalian Diaphanous 1 (mDia1) containing the GBD/FH3 region, an all-helical structure with armadillo repeats. Rho uses its ‘switch’ regions for interacting with two subdomains of GBD/FH3. We show that the FH3 domain of mDia1 forms a stable dimer and we also identify the DAD-binding site. Although binding of Rho and DAD on the N-terminal fragment of mDia1 are mutually exclusive, their binding sites are only partially overlapping. On the basis of our results, we propose a structural model for the regulation of mDia1 by Rho and DAD.

The regulation of mDia1 by autoinhibition and its release by Rho*GTP.

Formins induce the nucleation and polymerisation of unbranched actin filaments via the formin‐homology domains 1 and 2. Diaphanous‐related formins (Drfs) are regulated by a RhoGTPase‐binding domain situated in the amino‐terminal (N‐terminal) region and a carboxy‐terminal Diaphanous‐autoregulatory domain (DAD), whose interaction stabilises an autoinhibited inactive conformation. Binding of active Rho releases DAD and activates the catalytic activity of mDia. Here, we report on the interaction of DAD with the regulatory N‐terminus of mDia1 (mDiaN) and its release by Rho•GTP. We have defined the elements required for tight binding and solved the three‐dimensional structure of a complex between an mDiaN construct and DAD by X‐ray crystallography. The core DAD region is an α‐helical peptide, which binds in the most highly conserved region of mDiaN using mainly hydrophobic interactions. The structure suggests a two‐step mechanism for release of autoinhibition whereby Rho•GTP, although having a partially nonoverlapping binding site, displaces DAD by ionic repulsion and steric clashes. We show that Rho•GTP accelerates the dissociation of DAD from the mDiaN•DAD complex.

Specificity of Interactions between mDia Isoforms and Rho Proteins

Formins are key regulators of actin nucleation and polymerization. They contain formin homology 1 (FH1) and 2 (FH2) domains as the catalytic machinery for the formation of linear actin cables. A subclass of formins constitutes the Diaphanous-related formins, members of which are regulated by the binding of a small GTP-binding protein of the Rho subfamily. Binding of these molecular switch proteins to the regulatory N-terminal mDiaN, including the GTPase-binding domain, leads to the release of auto-inhibition. From the three mDia isoforms, mDia1 is activated only by Rho (RhoA, -B, and -C), in contrast to mDia2 and -3, which is also activated by Rac and Cdc42. Little is known about the determinants of specificity. Here we report on the interactions of RhoA, Rac1, and Cdc42 with mDia1 and an mDia1 mutant (mDiaN-Thr-Ser-His (TSH)), which based on structural information should mimic mDia2 and -3. Specificity is analyzed by biochemical studies and a structural analysis of a complex between Cdc42·Gpp(NH)p and mDiaN-TSH. A triple NNN motif in mDia1 (amino acids 164-166), corresponding to the TSH motif in mDia2/3 (amino acids 183-185 and 190-192), and the epitope interacting with the Rho insert helix are essential for high affinity binding. The triple N motif of mDia1 allows tight interaction with Rho because of the presence of Phe-106, whereas the corresponding His-104 in Rac and Cdc42 forms a complementary interface with the TSH motif in mDia2/3. We also show that the F106H and H104F mutations drastically alter the affinities and thermodynamics of mDia interactions.

Active site remodeling switches HIV specificity of antiretroviral TRIMCyp

TRIMCyps are primate antiretroviral proteins that potently inhibit HIV replication. Here we describe how rhesus macaque TRIMCyp (RhTC) has evolved to target and restrict HIV-2. We show that the ancestral cyclophilin A (CypA) domain of RhTC targets HIV-2 capsid with weak affinity, which is strongly increased in RhTC by two mutations (D66N and R69H) at the expense of HIV-1 binding. These mutations disrupt a constraining intramolecular interaction in CypA, triggering the complete restructuring (>16 Å) of an active site loop. This new configuration discriminates between divergent HIV-1 and HIV-2 loop conformations mediated by capsid residue 88. Viral sensitivity to RhTC restriction can be conferred or abolished by mutating position 88. Furthermore, position 88 determines the susceptibility of naturally occurring HIV-1 sequences to restriction. Our results reveal the complex molecular, structural and thermodynamic changes that underlie the ongoing evolutionary race between virus and host.

Acetylation regulates Cyclophilin A catalysis, immunosuppression and HIV isomerization

The detailed characterization of endogenous proteins and use of non-natural amino acid engineering allows the identification and structural and functional analysis of a post-translational modification in regulating ligand binding and enzyme activity. Supplementary information The online version of this article (doi:10.1038/nchembio.342) contains supplementary material, which is available to authorized users.

Small GTP-binding protein Ran is regulated by posttranslational lysine acetylation

Significance The small GTPase Ran plays fundamental roles in cellular processes such as nucleo-cytoplasmic transport, mitotic spindle formation, and nuclear envelope assembly. Recently, Ran was found to be lysine acetylated, among others, in functionally important regions such as switch I and switch II. Using the genetic code expansion concept we show that lysine acetylation affects many important aspects of Ran function such as RCC1-catalyzed nucleotide exchange, intrinsic nucleotide hydrolysis, import/export complex formation, and Ran subcellular localization. Finally, we present evidence for a regulation of Ran acetylation by sirtuin deacetylases and lysine acetyltransferases. Ran is a small GTP-binding protein of the Ras superfamily regulating fundamental cellular processes: nucleo-cytoplasmic transport, nuclear envelope formation and mitotic spindle assembly. An intracellular Ran•GTP/Ran•GDP gradient created by the distinct subcellular localization of its regulators RCC1 and RanGAP mediates many of its cellular effects. Recent proteomic screens identified five Ran lysine acetylation sites in human and eleven sites in mouse/rat tissues. Some of these sites are located in functionally highly important regions such as switch I and switch II. Here, we show that lysine acetylation interferes with essential aspects of Ran function: nucleotide exchange and hydrolysis, subcellular Ran localization, GTP hydrolysis, and the interaction with import and export receptors. Deacetylation activity of certain sirtuins was detected for two Ran acetylation sites in vitro. Moreover, Ran was acetylated by CBP/p300 and Tip60 in vitro and on transferase overexpression in vivo. Overall, this study addresses many important challenges of the acetylome field, which will be discussed.

Structural and mechanistic insights into the regulation of the fundamental Rho-regulator RhoGDIα by lysine acetylation

Rho proteins are small GTP/GDP-binding proteins primarily involved in cytoskeleton regulation. Their GTP/GDP cycle is often tightly connected to a membrane/cytosol cycle regulated by the Rho guanine nucleotide dissociation inhibitor α (RhoGDIα). RhoGDIα has been regarded as a housekeeping regulator essential to control homeostasis of Rho proteins. Recent proteomic screens showed that RhoGDIα is extensively lysine-acetylated. Here, we present the first comprehensive structural and mechanistic study to show how RhoGDIα function is regulated by lysine acetylation. We discover that lysine acetylation impairs Rho protein binding and increases guanine nucleotide exchange factor-catalyzed nucleotide exchange on RhoA, these two functions being prerequisites to constitute a bona fide GDI displacement factor. RhoGDIα acetylation interferes with Rho signaling, resulting in alteration of cellular filamentous actin. Finally, we discover that RhoGDIα is endogenously acetylated in mammalian cells, and we identify CBP, p300, and pCAF as RhoGDIα-acetyltransferases and Sirt2 and HDAC6 as specific deacetylases, showing the biological significance of this post-translational modification.

Insights into lysine-deacetylation of natively folded substrate proteins by sirtuins

Sirtuins are NAD+-dependent lysine deacylases, regulating a variety of cellular processes. The nuclear Sirt1, the cytosolic Sirt2, and the mitochondrial Sirt3 are robust deacetylases, whereas the other sirtuins have preferences for longer acyl chains. Most previous studies investigated sirtuin-catalyzed deacylation on peptide substrates only. We used the genetic code expansion concept to produce natively folded, site-specific, and lysine-acetylated Sirt1–3 substrate proteins, namely Ras-related nuclear, p53, PEPCK1, superoxide dismutase, cyclophilin D, and Hsp10, and analyzed the deacetylation reaction. Some acetylated proteins such as Ras-related nuclear, p53, and Hsp10 were robustly deacetylated by Sirt1–3. However, other reported sirtuin substrate proteins such as cyclophilin D, superoxide dismutase, and PEPCK1 were not deacetylated. Using a structural and functional approach, we describe the ability of Sirt1–3 to deacetylate two adjacent acetylated lysine residues. The dynamics of this process have implications for the lifetime of acetyl modifications on di-lysine acetylation sites and thus constitute a new mechanism for the regulation of proteins by acetylation. Our studies support that, besides the primary sequence context, the protein structure is a major determinant of sirtuin substrate specificity.

RhoGDIα Acetylation at K127 and K141 Affects Binding toward Nonprenylated RhoA

Rho proteins are major regulators of the cytoskeleton. As most Ras-related proteins, they switch between an active, GTP-bound and an inactive, GDP-bound conformation. Rho proteins are targeted to the plasma membrane via a polybasic region and a prenyl group attached to a C-terminal cysteine residue. To distribute Rho proteins in the cell, the molecular chaperone RhoGDIα binds to the prenylated Rho proteins forming a cytosolic pool of mainly GDP-loaded Rho. Most studies characterized the interaction of prenylated Rho proteins and RhoGDIα. However, RhoGDIα was also shown to bind to nonprenylated Rho proteins with physiologically relevant micomolar affinities. Recently, it was discovered that RhoGDIα is targeted by post-translational lysine acetylation. For one site, K141, it was hypothesized that acetylation might lead to increased levels of formation of filamentous actin and filopodia in mammalian cells. The functional consequences of lysine acetylation for the interplay with nonprenylated RhoA have not been investigated. Here, we report that lysine acetylation at lysines K127 and K141 in the RhoGDIα immunoglobulin domain interferes with the interaction toward nonprenylated RhoA using a combined biochemical and biophysical approach. We determined the first crystal structure of a doubly acetylated protein, RhoGDIα, in complex with RhoA·GDP. We discover that the C-terminus of RhoA adopts a different conformation forming an intermolecular β-sheet with the RhoGDIα immunoglobulin domain.

Lysine-acetylation as a fundamental regulator of Ran function: Implications for signaling of proteins of the Ras-superfamily.

The small GTP-binding protein Ran is involved in the regulation of essential cellular processes in interphase but also in mitotic cells: Ran controls the nucleocytoplasmic transport of proteins and RNA, it regulates mitotic spindle formation and nuclear envelope assembly. Deregulations in Ran dependent processes were implicated in the development of severe diseases such as cancer and neurodegenerative disorders. To understand how Ran-function is regulated is therefore of highest importance. Recently, several lysine-acetylation sites in Ran were identified by quantitative mass-spectrometry, some being located in highly important regions such as the P-loop, switch I, switch II and the G5/SAK motif. We recently reported that lysine-acetylation regulates nearly all aspects of Ran-function such as RCC1 catalyzed nucleotide exchange, intrinsic nucleotide hydrolysis, its interaction with NTF2 and the formation of import- and export-complexes. As a hint for its biological importance, we identified Ran-specific lysine-deacetylases (KDACs) and -acetyltransferases (KATs). Also for other small GTPases such as Ras, Rho, Cdc42, and for many effectors and regulators thereof, lysine-acetylation sites were discovered. However, the functional impact of lysine-acetylation as a regulator of protein function has only been marginally investigated so far. We will discuss recent findings of lysine-acetylation as a novel modification to regulate Ras-protein signaling.