Dynamic Solution Structures of Whole Human NAP1 Dimer Bound to One and Two Histone H2A-H2B Heterodimers Obtained by Integrative Methods.

Nucleosome assembly protein 1 (NAP1) binds to histone H2A-H2B heterodimers, mediating their deposition on and eviction from the nucleosome. Human NAP1 (hNAP1) consists of a dimerization core domain and intrinsically disordered C-terminal acidic domain (CTAD), both of which are essential for H2A-H2B binding. Several structures of NAP1 proteins bound to H2A-H2B exhibit binding polymorphisms of the core domain, but the distinct structural roles of the core and CTAD domains remain elusive. Here, we have examined dynamic structures of the full-length hNAP1 dimer bound to one and two H2A-H2B heterodimers by integrative methods. Nuclear magnetic resonance (NMR) spectroscopy of full-length hNAP1 showed CTAD binding to H2A-H2B. Atomic force microscopy revealed that hNAP1 forms oligomers of tandem repeated dimers; therefore, we generated a stable dimeric hNAP1 mutant exhibiting the same H2A-H2B binding affinity as wild-type hNAP1. Size exclusion chromatography (SEC), multi-angle light scattering (MALS) and small angle X-ray scattering (SAXS), followed by modelling and molecular dynamics simulations, have been used to reveal the stepwise dynamic complex structures of hNAP1 binding to one and two H2A-H2B heterodimers. The first H2A-H2B dimer binds mainly to the core domain of hNAP1, while the second H2A-H2B binds dynamically to both CTADs. Based on our findings, we present a model of the eviction of H2A-H2B from nucleosomes by NAP1.

NAP1-Related Protein 1 (NRP1) has multiple interaction modes for chaperoning histones H2A-H2B.

Nucleosome Assembly Protein 1 (NAP1) family proteins are evolutionarily conserved histone chaperones that play important roles in diverse biological processes. In this study, we determined the crystal structure of Arabidopsis NAP1-Related Protein 1 (NRP1) complexed with H2A-H2B and uncovered a previously unknown interaction mechanism in histone chaperoning. Both in vitro binding and in vivo plant rescue assays proved that interaction mediated by the N-terminal α-helix (αN) domain is essential for NRP1 function. In addition, the C-terminal acidic domain (CTAD) of NRP1 binds to H2A-H2B through a conserved mode similar to other histone chaperones. We further extended previous knowledge of the NAP1-conserved earmuff domain by mapping the amino acids of NRP1 involved in association with H2A-H2B. Finally, we showed that H2A-H2B interactions mediated by αN, earmuff, and CTAD domains are all required for the effective chaperone activity of NRP1. Collectively, our results reveal multiple interaction modes of a NAP1 family histone chaperone and shed light on how histone chaperones shield H2A-H2B from nonspecific interaction with DNA.

The intrinsic stability of H2B-ubiquitylated nucleosomes and their in vitro assembly/disassembly by histone chaperone NAP1.

BACKGROUND: Apart the gene-regulatory functions as docking sites for histone 'readers', some histone modifications could directly affect nucleosome structure. The H2BK34-ubiquitylation deposited by MOF-MSL complex, increases nucleosome dynamics in vitro and promotes donation of one H2A/H2B dimer to histone acceptors. METHODS: We evaluated temperature-depended stability of H2BK34-ubiquitylated nucleosomes under 'physiological' ionic conditions in the presence or absence of histone acceptor, and examined assembly and disassembly of ubiquitylated nucleosomes in vitro by recombinant mouse NAP1. RESULTS: H2BK34ub modification is sufficient to promote selective eviction of only one H2A/H2B dimer independently of histone-binding agents. Despite the robust H2A/H2B dimer-displacement effect of mNAP1 with the H2BK34ub (but not unmodified) nucleosomes, NAP1 could assemble symmetrically- or asymmetrically ubiquitylated nucleosomes under 'physiological' conditions in vitro. CONCLUSIONS AND GENERAL SIGNIFICANCE: The increased mobility of one nucleosomal H2A/H2B dimer is an intrinsic nucleosome destabilizing property of H2BK34 ubiquitylation that has the intranucleosome bases. The ability of NAP to reasonably efficiently assemble H2BK34-ubiquitylated nucleosomes supposes a potential mechanism for deposition/distribution of H2BK34ub mark in the MOF-MSL independent manner (for example, during histone dimer exchange upon transcription elongation).

Structural Insights into ceNAP1 Chaperoning Activity toward ceH2A-H2B.

In eukaryotes, nucleosome assembly is crucial for genome integrity. The histone chaperone NAP1 plays an important role in histone folding, storage, and transport, as well as histone exchange and nucleosome assembly. At present, the molecular basis of these activities is not fully understood. We have solved high-resolution crystal structures of Caenorhabditis elegans NAP1 (ceNAP1) in complex with its cognate substrates: the C. elegans H2A-H2B dimer (ceH2A-H2B) and the H2A.Z-H2B dimer (ceH2A.Z-H2B). Our structural and biochemical data reveals the acidic concave surface is relevant to tetramerization, and uncovers how a ceNAP1 homodimer uses its concave surface to asymmetrically recognize a ceH2A-H2B or ceH2A.Z-H2B heterodimer. Intriguingly, an "acidic strip" within the concave surface of ceNAP1 is crucial for binding histones, including H2A-H2B, H3-H4, and histone variants. Thus, our results provide insight into the molecular mechanisms of NAP1 histone chaperone activity.

How Protein Binding Sensitizes the Nucleosome to Histone H3K56 Acetylation.

The nucleosome, the fundamental gene-packing unit comprising an octameric histone protein core wrapped with DNA, has a flexible structure that enables dynamic gene regulation mechanisms. Histone lysine acetylation at H3K56 removes a positive charge from the histone core where it interacts with the termini of the nucleosomal DNA and acts as a critical gene regulatory signal that is implicated in transcription initiation and elongation. The predominant proposal for the biophysical role of H3K56 acetylation (H3K56ac) is that weakened electrostatic interaction between DNA termini and the histone core results in facilitated opening and subsequent disassembly of the nucleosome. However, this effect alone is too weak to account for the strong coupling between H3K56ac and its regulatory outcomes. Here we utilized a semisynthetically modified nucleosome with H3K56ac in order to address this discrepancy. Based on the results, we propose an innovative mechanism by which the charge neutralization effect of H3K56ac is significantly amplified via protein binding. We employed three-color single-molecule fluorescence resonance energy transfer (smFRET) to monitor the opening rate of nucleosomal DNA termini induced by binding of histone chaperone Nap1. We observed an elevated opening rate upon H3K56ac by 5.9-fold, which is far larger than the 1.5-fold previously reported for the spontaneous opening dynamics in the absence of Nap1. Our proposed mechanism successfully reconciles this discrepancy because DNA opening for Nap1 binding must be larger than the average spontaneous opening. This is a novel mechanism that can explain how a small biophysical effect of histone acetylation results in a significant change in protein binding rate.

Characterization of Caenorhabditis elegans Nucleosome Assembly Protein 1 Uncovers the Role of Acidic Tails in Histone Binding.

Nucleosome assembly proteins (Naps) influence chromatin dynamics by directly binding to histones. Here we provide a comprehensive structural and biochemical analysis of a Nap protein from Caenorhabditis elegans (CeNap1). CeNap1 naturally lacks the acidic N-terminal tail and has a short C-terminal tail compared to many other Nap proteins. Comparison of CeNap1 with full length and tail-less constructs of Saccharomyces cerevisiae Nap1 uncovers the role of these tails in self-association, histone binding, and Nap competition with DNA for H2A-H2B. We find that the presence of tails influences the stoichiometry of H2A-H2B binding and is required to complete the interactions between H2A-H2B and DNA. The absolute stoichiometry of the Nap protein and H2A-H2B complex is 2:1 or 2:2, with only a very small population of higher-order oligomers occurring at 150 mM NaCl. We also show that H3-H4 binds differently than H2A-H2B and that an (H3-H4)2 tetramer can simultaneously bind two Nap2 protein homodimers.

Structure of an H1-Bound 6-Nucleosome Array Reveals an Untwisted Two-Start Chromatin Fiber Conformation.

Chromatin adopts a diversity of regular and irregular fiber structures in vitro and in vivo. However, how an array of nucleosomes folds into and switches between different fiber conformations is poorly understood. We report the 9.7 Å resolution crystal structure of a 6-nucleosome array bound to linker histone H1 determined under ionic conditions that favor incomplete chromatin condensation. The structure reveals a flat two-start helix with uniform nucleosomal stacking interfaces and a nucleosome packing density that is only half that of a twisted 30-nm fiber. Hydroxyl radical footprinting indicates that H1 binds the array in an on-dyad configuration resembling that observed for mononucleosomes. Biophysical, cryo-EM, and crosslinking data validate the crystal structure and reveal that a minor change in ionic environment shifts the conformational landscape to a more compact, twisted form. These findings provide insights into the structural plasticity of chromatin and suggest a possible assembly pathway for a 30-nm fiber.

Histone octamer rearranges to adapt to DNA unwrapping.

Nucleosomes, the basic units of chromatin, package and regulate expression of eukaryotic genomes. Although the structure of the intact nucleosome is well characterized, little is known about structures of partially unwrapped, transient intermediates. In this study, we present nine cryo-EM structures of distinct conformations of nucleosome and subnucleosome particles. These structures show that initial DNA breathing induces conformational changes in the histone octamer, particularly in histone H3, that propagate through the nucleosome and prevent symmetrical DNA opening. Rearrangements in the H2A-H2B dimer strengthen interaction with the unwrapping DNA and promote nucleosome stability. In agreement with this, cross-linked H2A-H2B that cannot accommodate unwrapping of the DNA is not stably maintained in the nucleosome. H2A-H2B release and DNA unwrapping occur simultaneously, indicating that DNA is essential in stabilizing the dimer in the nucleosome. Our structures reveal intrinsic nucleosomal plasticity that is required for nucleosome stability and might be exploited by extrinsic protein factors.

Biochemical and Biophysical Characterisation of Higher Oligomeric Structure of Rat Nucleosome Assembly Protein 1.

Nucleosome assembly protein 1 (NAP1) is a histone chaperone that exchanges histone H2A-H2B dimer from chromatin templates. Studies with yeast NAP1 (yNAP1) have revealed its existence as multiple oligomeric species in solution. Here, rat NAP1 (rNAP1), which is 98% identical to the human NAP1 (hNAP1) was used as a model to characterize the oligomeric structures of this protein in higher eukaryotes. Gel filtration chromatography and Dynamic light scattering of recombinant rNAP1 indicated that the protein exists as a complex mixture of multimeric species even at 500 mM ionic strength. The solution-state complexity remains unchanged even at higher ionic strengths. Equilibrium unfolding (ΔG 14.6 kcal mol- 1) shows that rNAP1, both dimeric and oligomeric, follow the two-state model of unfolding with no detectable intermediates. Homology modelling suggests that rat and yeast NAP1 share an overall similar structure with conserved domains. However, dissimilar substitutions like threonine and lysine with glycine in the ß-hairpin involved in oligomerization, possibly leads to the observed differences in the oligomerization propensity of the two proteins. Molecular dynamic simulation (MDS) of the two structures also revealed that rNAP1 dimer is more stable owing to the extensive hydrogen bonding in comparison to yNAP1. Further, in vitro kinase assay showed that the phosphorylation of rNAP1 favors oligomerization with no effect on its histone binding capacity. Our results clearly suggest that there are differences in the in-solution behavior of rNAP1 compared to yNAP1 which may have in vivo functional implications for the regulation of these complexes during chromatin assembly and rearrangement.

Redundant Functions for Nap1 and Chz1 in H2A.Z Deposition.

H2A.Z is a histone H2A variant that contributes to transcriptional regulation, DNA damage response and limits heterochromatin spreading. In Saccharomyces cerevisiae, H2A.Z is deposited by the SWR-C complex, which relies on several histone chaperones including Nap1 and Chz1 to deliver H2A.Z-H2B dimers to SWR-C. However, the mechanisms by which Nap1 and Chz1 cooperate to bind H2A.Z and their contribution to H2A.Z deposition in chromatin is not well understood. Using structural modeling and molecular dynamics simulations, we identify a series of H2A.Z residues that form a chaperone-specific binding surface. Mutation of these residues revealed different surface requirements for Nap1 and Chz1 interaction with H2A.Z. Consistent with this result, we found that loss of Nap1 or Chz1 individually resulted in mild defects in H2A.Z deposition, but that deletion of both Nap1 and Chz1 resulted in a significant reduction of H2A.Z deposition at promoters and led to heterochromatin spreading. Together, our findings reveal unique H2A.Z surface dependences for Nap1 and Chz1 and a redundant role for these chaperones in H2A.Z deposition.

Single-Molecule Investigations on Histone H2A-H2B Dynamics in the Nucleosome.

Nucleosomes impose physical barriers to DNA-templated processes, playing important roles in eukaryotic gene regulation. DNA is packaged into nucleosomes by histone proteins mainly through strong electrostatic interactions that can be modulated by various post-translational histone modifications. Investigating the dynamics of histone dissociation from the nucleosome and how it is altered upon histone modifications is important for understanding eukaryotic gene regulation mechanisms. In particular, histone H2A-H2B dimer displacement in the nucleosome is one of the most important and earliest steps of histone dissociation. Two conflicting hypotheses on the requirement for dimer displacement are that nucleosomal DNA needs to be unwrapped before a dimer can displace and that a dimer can displace without DNA unwrapping. In order to test the hypotheses, we employed three-color single-molecule FRET and monitored in a time-resolved manner the early kinetics of H2A-H2B dimer dissociation triggered by high salt concentration and by histone chaperone Nap1. The results reveal that dimer displacement requires DNA unwrapping in the vast majority of the nucleosomes in the salt-induced case, while dimer displacement precedes DNA unwrapping in >60% of the nucleosomes in the Nap1-mediated case. We also found that acetylation at histone H4K16 or H3K56 affects the kinetics of Nap1-mediated dimer dissociation and facilitates the process both kinetically and thermodynamically. On the basis of these results, we suggest a mechanism by which histone chaperone facilitates H2A-H2B dimer displacement from the histone core without requiring another factor to unwrap the nucleosomal DNA.

KSHV encoded LANA recruits Nucleosome Assembly Protein NAP1L1 for regulating viral DNA replication and transcription.

The establishment of latency is an essential for lifelong persistence and pathogenesis of Kaposi's sarcoma-associated herpesvirus (KSHV). Latency-associated nuclear antigen (LANA) is the most abundantly expressed protein during latency and is important for viral genome replication and transcription. Replication-coupled nucleosome assembly is a major step in packaging the newly synthesized DNA into chromatin, but the mechanism of KSHV genome chromatinization post-replication is not understood. Here, we show that nucleosome assembly protein 1-like protein 1 (NAP1L1) associates with LANA. Our binding assays revealed an association of LANA with NAP1L1 in KSHV-infected cells, which binds through its amino terminal domain. Association of these proteins confirmed their localization in specific nuclear compartments of the infected cells. Chromatin immunoprecipitation assays from NAP1L1-depleted cells showed LANA-mediated recruitment of NAP1L1 at the terminal repeat (TR) region of the viral genome. Presence of NAP1L1 stimulated LANA-mediated DNA replication and persistence of a TR-containing plasmid. Depletion of NAP1L1 led to a reduced nucleosome positioning on the viral genome. Furthermore, depletion of NAP1L1 increased the transcription of viral lytic genes and overexpression decreased the promoter activities of LANA-regulated genes. These results confirmed that LANA recruitment of NAP1L1 helps in assembling nucleosome for the chromatinization of newly synthesized viral DNA.

Coordinated Action of Nap1 and RSC in Disassembly of Tandem Nucleosomes.

The SWI/SNF and RSC family of ATP-dependent chromatin remodelers disassembles nucleosomes by moving nucleosomes into the vicinity of adjoining nucleosomes. We found that the histone chaperone Nap1 efficiently promotes disassembly of adjacent nucleosomes with which RSC collides and not the disassembly of nucleosomes mobilized by RSC. Nap1 is specific to RSC, as it does not target SWI/SNF, its paralog in Saccharomyces cerevisiae Extensive mutational analysis of Nap1 has revealed that Nap1 affinity for histones H2A-H2B and H3-H4 and its ability to displace histones from DNA are required for Nap1 to enhance RSC-mediated disassembly. Other histone chaperones, such as Vps75, that also bind histones are not able to enhance RSC-mediated disassembly. Our study suggests a mechanism by which Nap1 is recruited to actively transcribed regions and assists in the passage of the transcription complex through chromatin, and it provides a novel mechanism for the coordinated action of RSC and Nap1.

Structural evidence for Nap1-dependent H2A-H2B deposition and nucleosome assembly.

Nap1 is a histone chaperone involved in the nuclear import of H2A-H2B and nucleosome assembly. Here, we report the crystal structure of Nap1 bound to H2A-H2B together with in vitro and in vivo functional studies that elucidate the principles underlying Nap1-mediated H2A-H2B chaperoning and nucleosome assembly. A Nap1 dimer provides an acidic binding surface and asymmetrically engages a single H2A-H2B heterodimer. Oligomerization of the Nap1-H2A-H2B complex results in burial of surfaces required for deposition of H2A-H2B into nucleosomes. Chromatin immunoprecipitation-exonuclease (ChIP-exo) analysis shows that Nap1 is required for H2A-H2B deposition across the genome. Mutants that interfere with Nap1 oligomerization exhibit severe nucleosome assembly defects showing that oligomerization is essential for the chaperone function. These findings establish the molecular basis for Nap1-mediated H2A-H2B deposition and nucleosome assembly.

Single-Molecule Studies of the Linker Histone H1 Binding to DNA and the Nucleosome.

Linker histone H1 regulates chromatin structure and gene expression. Investigating the dynamics and stoichiometry of binding of H1 to DNA and the nucleosome is crucial to elucidating its functions. Because of the abundant positive charges and the strong self-affinity of H1, quantitative in vitro studies of its binding to DNA and the nucleosome have generated results that vary widely and, therefore, should be interpreted in a system specific manner. We sought to overcome this limitation by developing a specially passivated microscope slide surface to monitor binding of H1 to DNA and the nucleosome at a single-molecule level. According to our measurements, the stoichiometry of binding of H1 to DNA and the nucleosome is very heterogeneous with a wide distribution whose averages are in reasonable agreement with previously published values. Our study also revealed that H1 does not dissociate from DNA or the nucleosome on a time scale of tens of minutes. We found that histone chaperone Nap1 readily dissociates H1 from DNA and superstoichiometrically bound H1 from the nucleosome, supporting a hypothesis whereby histone chaperones contribute to the regulation of the H1 profile in chromatin.

Comparing the Assembly and Handedness Dynamics of (H3.3-H4)2 Tetrasomes to Canonical Tetrasomes.

Eukaryotic nucleosomes consists of an (H3-H4)2 tetramer and two H2A-H2B dimers, around which 147 bp of DNA are wrapped in 1.7 left-handed helical turns. During chromatin assembly, the (H3-H4)2 tetramer binds first, forming a tetrasome that likely constitutes an important intermediate during ongoing transcription. We recently showed that (H3-H4)2 tetrasomes spontaneously switch between a left- and right-handed wrapped state of the DNA, a phenomenon that may serve to buffer changes in DNA torque induced by RNA polymerase in transcription. Within nucleosomes of actively transcribed genes, however, canonical H3 is progressively replaced by its variant H3.3. Consequently, one may ask if and how the DNA chirality dynamics of tetrasomes is altered by H3.3. Recent findings that H3.3-containing nucleosomes result in less stable and less condensed chromatin further underline the need to study the microscopic underpinnings of H3.3-containing tetrasomes and nucleosomes. Here we report real-time single-molecule studies of (H3.3-H4)2 tetrasome dynamics using Freely Orbiting Magnetic Tweezers and Electromagnetic Torque Tweezers. We find that the assembly of H3.3-containing tetrasomes and nucleosomes by the histone chaperone Nucleosome Assembly Protein 1 (NAP1) occurs in an identical manner to that of H3-containing tetrasomes and nucleosomes. Likewise, the flipping behavior of DNA handedness in tetrasomes is not impacted by the presence of H3.3. We also examine the effect of free NAP1, H3.3, and H4 in solution on flipping behavior and conclude that the probability for a tetrasome to occupy the left-handed state is only slightly enhanced by the presence of free protein. These data demonstrate that the incorporation of H3.3 does not alter the structural dynamics of tetrasomes, and hence that the preferred incorporation of this histone variant in transcriptionally active regions does not result from its enhanced ability to accommodate torsional stress, but rather may be linked to specific chaperone or remodeler requirements or communication with the nuclear environment.

Nucleosome assembly dynamics involve spontaneous fluctuations in the handedness of tetrasomes.

DNA wrapping around histone octamers generates nucleosomes, the basic compaction unit of eukaryotic chromatin. Nucleosome stability is carefully tuned to maintain DNA accessibility in transcription, replication, and repair. Using freely orbiting magnetic tweezers, which measure the twist and length of single DNA molecules, we monitor the real-time loading of tetramers or complete histone octamers onto DNA by Nucleosome Assembly Protein-1 (NAP1). Remarkably, we find that tetrasomes exhibit spontaneous flipping between a preferentially occupied left-handed state (ΔLk = -0.73) and a right-handed state (ΔLk = +1.0), separated by a free energy difference of 2.3 kBT (1.5 kcal/mol). This flipping occurs without concomitant changes in DNA end-to-end length. The application of weak positive torque converts left-handed tetrasomes into right-handed tetrasomes, whereas nucleosomes display more gradual conformational changes. Our findings reveal unexpected dynamical rearrangements of the nucleosomal structure, suggesting that chromatin can serve as a "twist reservoir," offering a mechanistic explanation for the regulation of DNA supercoiling in chromatin.

Histone chaperone-mediated nucleosome assembly process.

A huge amount of information is stored in genomic DNA and this stored information resides inside the nucleus with the aid of chromosomal condensation factors. It has been reported that the repeat nucleosome core particle (NCP) consists of 147-bp of DNA and two copies of H2A, H2B, H3 and H4. Regulation of chromosomal structure is important to many processes inside the cell. In vivo, a group of histone chaperones facilitate and regulate nucleosome assembly. How NCPs are constructed with the aid of histone chaperones remains unclear. In this study, the histone chaperone-mediated nucleosome assembly process was investigated using single-molecule tethered particle motion (TPM) experiments. It was found that Asf1 is able to exert more influence than Nap1 and poly glutamate acid (PGA) on the nucleosome formation process, which highlights Asf1's specific role in tetrasome formation. Thermodynamic parameters supported a model whereby energetically favored nucleosomal complexes compete with non-nucleosomal complexes. In addition, our kinetic findings propose the model that histone chaperones mediate nucleosome assembly along a path that leads to enthalpy-favored products with free histones as reaction substrates.

The histone chaperones Vps75 and Nap1 form ring-like, tetrameric structures in solution.

NAP-1 fold histone chaperones play an important role in escorting histones to and from sites of nucleosome assembly and disassembly. The two NAP-1 fold histone chaperones in budding yeast, Vps75 and Nap1, have previously been crystalized in a characteristic homodimeric conformation. In this study, a combination of small angle X-ray scattering, multi angle light scattering and pulsed electron-electron double resonance approaches were used to show that both Vps75 and Nap1 adopt ring-shaped tetrameric conformations in solution. This suggests that the formation of homotetramers is a common feature of NAP-1 fold histone chaperones. The tetramerisation of NAP-1 fold histone chaperones may act to shield acidic surfaces in the absence of histone cargo thus providing a 'self-chaperoning' type mechanism.

Phosphoregulation of Nap1 plays a role in septin ring dynamics and morphogenesis in Candida albicans.

UNLABELLED: Nap1 has long been identified as a potential septin regulator in yeasts. However, its function and regulation remain poorly defined. Here, we report functional characterization of Nap1 in the human-pathogenic fungus Candida albicans. We find that deletion of NAP1 causes constitutive filamentous growth and changes of septin dynamics. We present evidence that Nap1's cellular localization and function are regulated by phosphorylation. Phos-tag gel electrophoresis revealed that Nap1 phosphorylation is cell cycle dependent, exhibiting the lowest level around the time of bud emergence. Mass spectrometry identified 10 phosphoserine and phosphothreonine residues in a cluster near the N terminus, and mutation of these residues affected Nap1's localization to the septin ring and cellular function. Nap1 phosphorylation involves two septin ring-associated kinases, Cla4 and Gin4, and its dephosphorylation occurs at the septin ring in a manner dependent on the phosphatases PP2A and Cdc14. Furthermore, the nap1Δ/Δ mutant and alleles carrying mutations of the phosphorylation sites exhibited greatly reduced virulence in a mouse model of systemic candidiasis. Together, our findings not only provide new mechanistic insights into Nap1's function and regulation but also suggest the potential to target Nap1 in future therapeutic design. IMPORTANCE: Septins are conserved filament-forming GTPases involved in a wide range of cellular events, such as cytokinesis, exocytosis, and morphogenesis. In Candida albicans, the most prevalent human fungal pathogen, septin functions are indispensable for its virulence. However, the molecular mechanisms by which septin structures are regulated are poorly understood. In this study, we deleted NAP1, a gene encoding a putative septin regulator, in C. albicans and found that cells lacking NAP1 showed abnormalities in morphology, invasive growth, and septin ring dynamics. We identified a conserved N-terminal phosphorylation cluster on Nap1 and demonstrated that phosphorylation at these sites regulates Nap1 localization and function. Importantly, deletion of NAP1 or mutation in the N-terminal phosphorylation cluster strongly reduced the virulence of C. albicans in a mouse model of systemic infection. Thus, this study not only provides mechanistic insights into septin regulation but also suggests Nap1 as a potential antifungal target.